Co-crosslinked hyaluronic acid-silk fibroin hydrogels for improving tissue graft viability and for soft tissue augmentation

ABSTRACT

Hydrogels comprising a macromolecular matrix and water may be used to augment soft tissue of a human being, promote or support cell or tissue viability or proliferation, create space in tissue, and for other purposes. A macromolecular matrix may comprise a hyaluronic acid component crosslinked to a silk fibroin component.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/686,036, filed on Aug. 24, 2017, which claims priority to and thebenefit of U.S. Provisional Patent Application Ser. No. 62/379,045,filed Aug. 24, 2016, each of which is incorporated herein by referencein their entirety.

BACKGROUND Field of the Inventions

The present disclosure generally relates to crosslinked silk-hyaluronicacid compositions, methods of making and uses thereof, and morespecifically relates to silk-hyaluronic acid compositions useful forimproving tissue graft viability and soft tissue augmentation.

Description of the Related Art

Autologous fat transfer (“AFT”), also known as fat grafting, is aprocess by which fat is harvested from one part of a human body andinjected into another part of the same person's body where additionalbulk may be needed for cosmetic and/or aesthetic purposes. Clinicalapplications for autologous fat transfer are expanding rapidly withrecent reported use in breast reconstruction and augmentation, buttockenhancement, treatment of congenital tissue defects, facialreconstruction, and skin rejuvenation. Although this is a veryattractive approach and there is an increased trend in replacement ofsoft tissue volume with AFT, typical survival rates of grafted fat maybe poor and overall results may not be satisfactory to a patient.

AFT has been shown to be enhanced by the inclusion of hydrogels or otherscaffolds for tissue engineering. U.S. Pat. No. 9,662,422 to Pollock etal.; Crosslinked hyaluronic acid-collagen gels for improving tissuegraft viability and soft tissue augmentation; describes the use of ahyaluronic acid-collagen hydrogel in AFT. U.S. Patent Application Pub.No. 2013/0244943 A1: Yu et al.; Hyaluronic acid-collagen matrices fordermal filling and volumizing applications; describes the production ofcross-linked hyaluronic acid and collagen compositions. U.S. Pat. No.9,408,797 to Nijkang et al.; Dermal filler compositions for fine linetreatment; describes the use of a dermal filler comprising hyaluronicacid crosslinked with collagen in the treatment of facial wrinkles. Eachof these references is herein incorporated by reference in theirentirities.

Hyaluronic acid (HA) (synonymously “hyaluron” or “hyaluronate”) is anaturally occurring glycosaminoglycan that has been used as aconstituent of a dermal filler for wrinkle reduction and tissuevolumizing. Hyaluronan is an anionic, nonsulfated glycosaminoglycandistributed widely throughout connective, epithelial, and neuraltissues. Polymeric hyaluronic acid can have a molecular weight ofseveral million Daltons. A person typically has about 15 grams ofhyaluronan in his body about a third of which every day is degraded byendogenous enzymes and free radicals within a few hours or days andreplaced by hyaluronic acid newly synthesized by the body.

Silk is a natural (non-synthetic) protein made of high strength fibroinfibers with mechanical properties similar to or better than many ofsynthetic high performance fibers. Silk is also stable at physiologicaltemperatures in a wide range of pH, and is insoluble in most aqueous andorganic solvents. As a protein, unlike the case with most if not allsynthetic polymers, the degradation products (e.g., peptides, aminoacids) of silk are biocompatible. Silk is non-mammalian derived andcarries far less bioburden than other comparable natural biomaterials(e.g., bovine or porcine derived collagen). Silk, as the term isgenerally known in the art, means a filamentous fiber product secretedby an organism such as a silkworm or spider. Silks can be made bycertain insects such as for example Bombyx mori silkworms, and Nephiliaclavipes spiders. There are many variants of natural silk. Fibroin isproduced and secreted by a silkworm's two silk glands. As fibroin leavesthe glands it is coated with sericin a glue-like substance. Spider silkis produced as a single filament lacking the immunogenic proteinsericin.

Silk has been used in biomedical applications. The Bombyx mori speciesof silkworm produces a silk fiber (a “bave”) and uses the fiber to buildits cocoon. The bave as produced include two fibroin filaments orbroins, which are surrounded with a coating of the gummy, antigenicprotein sericin. Silk fibers harvested for making textiles, sutures andclothing are not sericin extracted or are sericin depleted or only to aminor extent and typically the silk remains at least 10% to 26% byweight sericin. Retaining the sericin coating protects the frail fibroinfilaments from fraying during textile manufacture. Hence textile gradesilk is generally made of sericin coated silk fibroin fibers. Medicalgrade silkworm silk is used as either as virgin silk suture, where thesericin has not been removed, or as a silk suture from which the sericinhas been removed and replaced with a wax or silicone coating to providea barrier between the silk fibroin and the body tissue and cells. Thusthere is a need for a sericin extracted implantable, bioresorbable silkdevice that promotes ingrowth of cells.

Bioconjugate Chemistry, 2010, 21, 240-247: Joem Y., et al., Effect ofcross-linking reagents for hyaluronic acid hydrogel dermal fillers ontissue augmentation and regeneration, discusses use of a particularcross-linked HMDA to prepare a cross-linked hyaluronic acid dermalfiller, and also discloses use of a variety of hyaluronic acid crosslinkers and hyaluronic activators including BDDE and EDC. CarbohydratePolymers, 2007, 70, 251-257: Jeon, O., et al., Mechanical properties anddegradation behaviors of hyaluronic acid hydrogels cross-linked atvarious cross-linking densities, discusses properties of hyaluronic acidcross linked with a polyethylene glycol diamine (a PEG-diamine). J. Am.Chem. Soc., 1955, 77 (14), 3908-3913: Schroeder W., et al., The aminoacid composition of Bombyx mori silk fibroin and of Tussah silk fibroin,compares the amino acid compositions of the silk from two silkwormspecies. U.S. Patent Application Pub. No. US 2010/0016886 A1: Lu, H.,High swell, long lived hydrogel sealant; discusses reacting a multi-armamine (i.e., an 9 arm polyethelene glycol (PEG) with an oxidized (i.e.,to introduce aldehyde groups) polysaccharide (such as hyaluronic acid),useful for tissue augmentation or a tissue adhesive/sealant. U.S. Pat.No. 6,903,199 to Moon. T., et al., Crosslinked amide derivatives ofhyaluronic acid and manufacturing method thereof discusses cross linkinghyaluronic acid with a chitosan or with a deacetylated hyaluronic acidwith reactive amide groups, using (for example) EDC or NHS. U.S. PatentApplication Pub. No. US 2016/0361247 A1: Pavlovic et al., Cross linkedsilk-hyaluronic acid composition; describes methods for cross-linkingsilk with hyaluronic acid. U.S. Pat. No. 8,288,347 to Collette et al.,Dermal fillers comprising silk fibroin hydrogels and uses thereofdescribes methods for purifying silk fibroins and hydrogels comprisingsilk fibroin with or without an amphiphilic peptide.

International Patent Application WO/2010/123945, Altman, G., et al.,Silk fibroin hydrogels and uses thereof discusses silk hydrogels madeby, for example, digesting degummed silk hydrogels made by, for example,digesting degummed Bombyx mori silk at 60° C. for 4 hours in 9.3Mlithium bromide to thereby obtain a 20% silk solution, an 8% silksolution of which was induced to gel using 23RGD and/or ethanol, whichcan be present in a hyaluronic acid carrier. Altman also discussespossible use as a dermal filler and to promote wound closure, and a silkhydrogel coating on a silk mesh. Altman also discusses silk cross linkedto hyaluronic acid (see paragraphs [213] to [220], using various crosslinkers.

International Patent Application. Pub. No. WO/2008/008857: Prestwich,G., et al., Tholated macromolecules and methods for making and usingthereof discloses a thioethyl ether substituted hyaluronic acid made byoxidating coupling useful, for example, in arthritis treatment.International Patent Application. Pub. No. WO/2008/008859: Prestwich,G., et al., Macromolecules modified with electrophilic groups andmethods of making and using thereof discloses a haloacetate derivativehyaluronic acid reacted with thiol modified hyaluronic acid to make ahydrogel, with various medical uses. Biomacromolecules, 2010, 11 (9),2230-2237: Serban, M., et. Al., Modular elastic patches: mechanical andbiological effects discusses how to make an elastic patch by crosslinking elastin, hyaluronic acid and silk, by adding an aminatedhyaluronic acid (made using EDC) with a 20% silk solution and elastin,in phosphate buffered saline (PBS) with BS3 (bissulfosuccinimidylsuberate, as cross linker) at 37° C. for 12 hours. Biomaterials, 2008,29(10), 1388-1399: Serban, M., et al., Synthesis, characterization andchondroprotective properties of a hyaluronan thioethyl ether derivativediscusses a viscous 2-thioethyl ether hyaluronic acid derivativesolution useful for viscosupplementation in arthritis treatment. Theabstract mentions that a prior hyaluronic acid with multiple thio groupscan be used for adhesion prevention. Methods, 2008, 45, 93-98: Serban,M., et al., Modular extracellular matrices: solutions to the puzzlediscusses cross linked thio modified hyaluronic acid hydrogel useful asa semi synthetic extracellular matrix for cell culture.Biomacromolecules, 2007, 8(9), 2821-2828: Serban, M., et al., Synthesisof hyaluronan haloacetates and biology of novel cross linker freesynthetic extracellular matrix hydrogels discusses cross linkinghaloacetate substituted hyaluronic acids reacted with a thiolsubstituted hyaluronic acid to make a hydrogel useful for cell cultureor adhesion prevention or medical device coating. Journal of MaterialsChemistry, 2009, 19, 6443-6450: Murphy A., et al., Biomedicalapplications of chemically modified silk fibroin is a review of methodsto make silk conjugates, including silk conjugated to oligosaccharides,modified silk and medical uses. Biomacromolecules, 2004, 5, 751-757:Sohn, S., et al., Phase behavior and hydration of silk fibroin discussesa study of Bombyx mori silk in vitro using osmotic stress, determiningthat silk I (α-silk) but not silk II (β-sheet, spun silk fiber) ishydrated. U.S. Pat. No. 8,071,722 to Kaplan, D., et al., SilkBiomaterials and methods of use thereof discloses silk films, use of9-12 m LiBr to dissolve extracted silk, adding hyaluronic acid to a silksolution to make fibers from the composition. See also eg the Kaplanpatents and application Ser. Nos. 7,674,882; 8,178,656; 2010 055438,and; 2011 223153. U.S. Patent application 2011 071239 by Kaplan, D., etal., PH induced silk gels and uses thereof discloses methods for makingsilk fibroin gel from silk fibroin solution, useful to coat a medicaldevice using implants, as an injectable gel to fill a tissue void,making an adhesive silk gel (with or without a hyaluronic acid),adhering the adhesive silk gel to a subject for example for use as awound bioadhesive, a multi-layered silk gel. U.S. Patent application2009 0202614 by Kaplan, D., et al., Methods for stepwise deposition ofsilk fibroin coatings discusses layered silk coatings, silk films madeusing silk fibroin solutions, which can include a hyaluronic acid,useful, for example, as wound healing patches, to coat an implantablemedical device. U.S. Pat. No. 4,818,291 to Iwatsuki M., et al.,Silk-fibroin and human-fibrinogen adhesive composition discussessurgical adhesive useful in tissue repair made as a mixture of LiBrdissolved silk and fibrinogen.

To increase in vivo residence time, the linear chains of hyaluronic acidcan be crosslinked with a small molecular cross linker such as, forexample, butanediol diglycidyl ether (BDDE) or1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)chemistry. Crosslinking hyaluronic acid with BDDE is usually carried outat high pH (>12) and at temperatures of about 50° C. It has beenreported that the degradation rate constant of HA is increased roughly100 times when the temperature and pH are both increased from 40 to 60°C. and 7 to 11 respectively.

SUMMARY

The present disclosure addresses these and other shortcomings in thefield of cosmetic and reconstructive medicine and procedures.

Hydrogels and hydrogel compositions have been developed that are usefulfor soft tissue augmentation procedures, including tissue reconstructionprocedures. These hydrogels and hydrogel compositions may promote and/orsupport the survival or growth of living cells and other components oftissues.

In some embodiments, a soft tissue augmentation product is provided,which can be injected or introduced into tissue along with a cellularcomponent. The product may comprise a forming component comprising ahydrogel described herein, the hydrogel having a form suitable foraugmenting human soft tissue by introducing, for example, by injectionor implantation, the forming component into the human tissue. In someembodiments, the hydrogel itself contains or includes a cellularmaterial, for example, living tissue or living cells, and a componenthyaluronic acid and silk fibroin. The product may further comprise alabel including instructions for such injecting or implanting theforming component. In addition, the product may, in some embodiments,include a syringe or other device for facilitating the introducing ofthe forming component.

Typically, in accordance with some embodiments, a hydrogel or a hydrogelcomposition may comprise water, and a crosslinked macromolecular matrix.The matrix may be in a form suitable for mixing or combining with livingcells or tissue prior to introduction of the matrix into the portion oranatomical feature being augmented. In some embodiments, the matrixcomprises a hyaluronic acid component; and a silk fibroin component. Insome embodiments, the hyaluronic acid is crosslinked to the silkfibroin, for example, by a crosslinking component. In one especiallyadvantageous embodiment, at least a portion of the crosslink units ofthe crosslinking component comprises an ester bond or an amide bond.

In some embodiments, methods of augmenting soft tissue of a human beingare provided, which comprise injecting or implanting a hydrogelcomposition described herein into a soft tissue of the human being tothereby augment the soft tissue. In some embodiments, the methodincludes combining, or mixing the hydrogel composition with living cellsor tissue that have been explanted from the patient. The composition maybe especially effective in enhancing cell proliferation and/orsupporting cell viability when reintroduced, for example, into a breastof a patient. Thus, the method in these instances may be useful inconjunction with fat grafting procedures.

Some embodiments are directed toward methods of promoting or supportingcell proliferation or survival, for example, in fat grafting proceduresor other augmentation or reconstructive procedures. For example, themethods may include contacting hydrogel compositions described hereinwith cellular materials, cells and/or tissue, for example, prior toinjecting the compositions into the body.

In some embodiments, methods are provides for preparing a space in humanor animal tissue, for example, for later receipt of a fat graft orimplant, the method comprising injecting a hydrogel compositiondescribed herein into the tissue, and allowing growth or proliferationof tissue while the composition degrades over time.

Additional features and advantages of the subject technology will be setforth in the description below, and in part will be apparent from thedescription, or may be learned by practice of the subject technology.The advantages of the subject technology will be realized and attainedby the structure particularly pointed out in the written description andembodiments hereof as well as the appended drawings.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the subject technology.

BRIEF DESCRIPTION OF THE DRAWINGS

Various features of illustrative embodiments of the inventions aredescribed below with reference to the drawings. The illustratedembodiments are intended to illustrate, but not to limit, theinventions. The drawings contain the following figures:

FIG. 1 provides a graph showing the in vivo volume retention over timefor hyalruonic acid-silk fibroin (HA-Fbn) hydrogels combined withlipoaspirate in comparison with a lipoaspirate-only control.

FIG. 2A provides a photograph of a tissue sample extracted after in vivofat grafting of a composition consisting of only lipoaspirate.

FIG. 2B provides a micrograph at 5× magnification of a tissue sampleextracted and stained after in vivo fat grafting of a compositionconsisting of only lipoaspirate.

FIG. 3A provides a photograph of a tissue sample extracted after in vivofat grafting of a hydrogel sample containing lipoaspirate in combinationwith a co-crosslinked HA-silk fibroin composition in a HA:silk fibroinratio of 19:2.

FIG. 3B provides a micrograph at 5× magnification of a tissue sampleextracted and stained after in vivo fat grafting of a hydrogel samplecontaining lipoaspirate in combination with a co-crosslinked HA-silkfibroin composition in a HA:silk fibroin ratio of 19:2.

FIG. 4A provides a photograph of a tissue sample extracted after in vivofat grafting of a hydrogel sample containing lipoaspirate in combinationwith a co-crosslinked HA-silk fibroin composition in a HA:silk fibroinratio of 18:3.

FIG. 4B provides a micrograph at 5× magnification of a tissue sampleextracted and stained after in vivo fat grafting of a hydrogel samplecontaining lipoaspirate in combination with a co-crosslinked HA-silkfibroin composition in a HA:silk fibroin ratio of 18:3.

FIG. 5A provides a photograph of a tissue sample extracted after in vivofat grafting of a hydrogel sample containing lipoaspirate in combinationwith a co-crosslinked HA-silk fibroin composition in a HA:silk fibroinratio of 17:4.

FIG. 5B provides a micrograph at 5× magnification of a tissue sampleextracted and stained after in vivo fat grafting of a hydrogel samplecontaining lipoaspirate in combination with a co-crosslinked HA-silkfibroin composition in a HA:silk fibroin ratio of 17:4.

DETAILED DESCRIPTION

It is understood that various configurations of the subject technologywill become readily apparent to those skilled in the art from thedisclosure, wherein various configurations of the subject technology areshown and described by way of illustration. As will be realized, thesubject technology is capable of other and different configurations andits several details are capable of modification in various otherrespects, all without departing from the scope of the subjecttechnology. Accordingly, the summary, drawings and detailed descriptionare to be regarded as illustrative in nature and not as restrictive.

The detailed description set forth below is intended as a description ofvarious configurations of the subject technology and is not intended torepresent the only configurations in which the subject technology may bepracticed. The appended drawings are incorporated herein and constitutea part of the detailed description. The detailed description includesspecific details for the purpose of providing a thorough understandingof the subject technology. However, it will be apparent to those skilledin the art that the subject technology may be practiced without thesespecific details. In some instances, well-known structures andcomponents are shown in block diagram form in order to avoid obscuringthe concepts of the subject technology. Like components are labeled withidentical element numbers for ease of understanding.

Hydrogels described herein may be used to augment soft tissue of a humanbeing. For example, a hydrogel or a hydrogel composition may be injectedor implanted a hydrogel composition into a soft tissue of the humanbeing to thereby augment the soft tissue. In some embodiments, a formingcomponent may comprise a hydrogel or a hydrogel having a form suitablefor augmenting human soft tissue by injecting or implanting the formingcomponent into the human tissue.

A forming component may be any object or substance with a form that issuitable for a particular augmentation need. For example, a formingcomponent may have a viscosity, firmness, and/or other physicalproperties, such that, when injected or implanted into a soft tissue toaugment the tissue, the newly augmented portion of the tissue isreasonably similar to the natural tissue. If a forming component is tobe injected, it may be in a form that is suitable for injection. Forexample, the viscosity may be low enough so that injection through aneedle is possible. If a forming component is to be implanted, in somecircumstances it may be desirable for the forming component to be solidor sufficiently viscous so as to maintain its shape during implantation.

Some augmentation products may include a label comprising instructionsto inject or implant the forming component into the human tissue.

Hydrogels described herein may also be used to enhance, promote orsupport cell proliferation or survival. Some embodiments include amethod comprising contacting a hydrogel or a hydrogel composition with acell or cells.

A hydrogel or a hydrogel composition that contacts one or more cells maypromote or support survival of the cells, including adypocytes,adipose-derived stem cells, stromal vascular fraction cells, or acombination thereof. For example, a hydrogel or a hydrogel compositiondescribed herein may promote or support cell survival to a greaterextent than a hydrogel composition comprising hyaluronic acid having aweight concentration that is similar to the weight concentration of thecrosslinked macromolecular matrix used in a hydrogel described herein.In some embodiments, a hydrogel or a hydrogel composition describedherein may promote or support cell survival to a greater extent than ahydrogel composition comprising water and hyaluronic acid at aconcentration of about 24 mg/mL or about 16 mg/mL. Contact between ahydrogel or a hydrogel composition described herein and cells maypromote or support cell survival in vivo to a greater extent than ahydrogel composition that is substantially identical except that thehyaluronic acid component and the silk fibroin component are notcrosslinked. In some embodiments, a hydrogel or a hydrogel compositionmay promote or support cell survival about as well as, or better than,tissue culture polystyrene.

A hydrogel composition disclosed herein may enhance survival of one ormore cells. In one embodiment, a hydrogel composition disclosed hereinenhances survival of one or more cells as compared to cells alone. Insome embodiments, a hydrogel composition disclosed herein enhancessurvival of one or more cells by at least about 50% at least about 100%,at least about 150%, at least about 200%, at least 250%, at least about300%, at least about 350%, at least about 400%, at least about 450%, orat least about 500% as compared to cells alone. In some embodiments, ahydrogel composition disclosed herein enhances survival of one or morecells by about 50% to about 250%, about 50% to about 500%, about 50% toabout 1000%, about 100% to about 300%, about 100% to about 500%, about150% to about 400%, about 150% to about 500%, or about 200% to about500%, as compared to cells alone.

In some embodiments, a hydrogel composition disclosed herein enhancessurvival of one or more cells as compared to cells with a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the silk fibroin component are not crosslinked. Insome embodiments, a hydrogel composition disclosed herein enhancessurvival of one or more cells by at least about 50% at least about 100%,at least about 150%, at least about 200%, at least 250%, at least about300%, at least about 350%, at least about 400%, at least about 450%, orat least about 500% as compared to cells with a hydrogel compositionthat is substantially identical except that the hyaluronic acidcomponent and the silk fibroin component are not crosslinked. In someembodiments, a hydrogel composition disclosed herein enhances survivalof one or more cells by about 50% to about 250%, about 50% to about500%, about 50% to about 1000%, about 100% to about 300%, about 100% toabout 500%, about 150% to about 400%, about 150% to about 500%, or about200% to about 500% as compared to cells with a hydrogel composition thatis substantially identical except that the hyaluronic acid component andthe silk fibroin component are not crosslinked. In some embodiments, ahydrogel composition that is substantially identical to a hydrogelcomposition disclosed herein except that the hyaluronic acid componentand the silk fibroin component are not crosslinked comprises hyaluronicacid at a concentration of about 16 mg/mL and water.

In yet another embodiment, a hydrogel composition disclosed hereinenhances survival of one or more cells as compared to cells with ahydrogel composition that is substantially identical except that thesilk fibroin component is absent. In some embodiments, a hydrogelcomposition disclosed herein enhances survival of one or more cells byat least about 50% at least about 100%, at least about 150%, at leastabout 200%, at least 250%, at least about 300%, at least about 350%, atleast about 400%, at least about 450%, or at least about 500% ascompared to cells with a hydrogel composition that is substantiallyidentical except that the silk fibroin component is absent. In someembodiments, a hydrogel composition disclosed herein enhances survivalof one or more cells by about 50% to about 250%, about 50% to about500%, about 50% to about 1000%, about 100% to about 300%, about 100% toabout 500%, about 150% to about 400%, about 150% to about 500%, or about200% to about 500% as compared to cells with a hydrogel composition thatis substantially identical except that the silk fibroin component isabsent. In some embodiments, a hydrogel composition that issubstantially identical to a hydrogel composition disclosed hereinexcept that the silk fibroin component is absent comprises hyaluronicacid at a concentration of about 16 mg/mL and water.

A hydrogel or a hydrogel composition that contacts one or more cells maypromote or support proliferation of cells, such as regenerative cells,stem cells, progenitor cells, precursor cells, adipose-derived stemcells, stromal vascular fraction cells, etc. A hydrogel or a hydrogelcomposition described herein may also promote or support cellproliferation to a greater extent than a hydrogel composition comprisinghyaluronic acid having a weight concentration that is similar to theweight concentration of the crosslinked macromolecular matrix used in ahydrogel described herein. In some embodiments, a hydrogel or a hydrogelcomposition described herein may promote or support cell proliferationto a greater extent than a hydrogel composition comprising water andhyaluronic acid at a concentration of about 24 mg/mL or about 16 mg/mL.Contact between a hydrogel or a hydrogel composition described hereinand cells may promote or support cell proliferation to a greater extentthan a hydrogel composition that is substantially identical except thatthe hyaluronic acid component and the silk fibroin component are notcrosslinked. In some embodiments, a hydrogel or a hydrogel compositionmay promote or support cell proliferation about as well as, or betterthan, tissue culture polystyrene.

A hydrogel composition disclosed herein may enhance proliferation of oneor more cells. In one embodiment, a hydrogel composition disclosedherein enhances proliferation of one or more cells as compared to cellsalone. In some embodiments, a hydrogel composition disclosed hereinenhances proliferation of one or more cells by at least about 50% atleast about 100%, at least about 150%, at least about 200%, at least250%, at least about 300%, at least about 350%, at least about 400%, atleast about 450%, or at least about 500 as compared to cells alone. Insome embodiments, a hydrogel composition disclosed herein enhancesproliferation of one or more cells by about 50% to about 250%, about 50%to about 500%, about 50% to about 1000%, about 100% to about 300%, about100% to about 500%, about 150% to about 400%, about 150% to about 500%,or about 200% to about 500%, as compared to cells alone.

In another embodiment, a hydrogel composition disclosed herein enhancesproliferation of one or more cells as compared to cells with a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the silk fibroin component are not crosslinked. Insome embodiments, a hydrogel composition disclosed herein enhancesproliferation of one or more cells by at least about 50% at least about100%, at least about 150%, at least about 200%, at least 250%, at leastabout 300%, at least about 350%, at least about 400%, at least about450%, or at least about 500% as compared to cells with a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the silk fibroin component are not crosslinked. Insome embodiments, a hydrogel composition disclosed herein enhancesproliferation of one or more cells by about 50% to about 250%, about 50%to about 500%, about 50% to about 1000%, about 100% to about 300%, about100% to about 500%, about 150% to about 400%, about 150% to about 500%,or about 200% to about 500% as compared to cells with a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the silk fibroin component are not crosslinked. Insome embodiments, a hydrogel composition that is substantially identicalto a hydrogel composition disclosed herein except that the hyaluronicacid component and the silk fibroin component are not crosslinkedcomprises hyaluronic acid at a concentration of about 16 mg/mL or about24 mg/mL and water.

In yet another embodiment, a hydrogel composition disclosed hereinenhances proliferation of one or more cells as compared to cells with ahydrogel composition that is substantially identical except that thesilk fibroin component is absent. In some embodiments, a hydrogelcomposition disclosed herein enhances proliferation of one or more cellsby at least about 50% at least about 100%, at least about 150%, at leastabout 200%, at least 250%, at least about 300%, at least about 350%, atleast about 400%, at least about 450%, or at least about 500% ascompared to cells with a hydrogel composition that is substantiallyidentical except that the silk fibroin component is absent. In someembodiments, a hydrogel composition disclosed herein enhancesproliferation of one or more cells by about 50% to about 250%, about 50%to about 500%, about 50% to about 1000%, about 100% to about 300%, about100% to about 500%, about 150% to about 400%, about 150% to about 500%,or about 200% to about 500% as compared to cells with a hydrogelcomposition that is substantially identical except that the silk fibroincomponent is absent. In some embodiments, a hydrogel composition that issubstantially identical to a hydrogel composition disclosed hereinexcept that the silk fibroin component is absent comprises hyaluronicacid at a concentration of about 16 mg/mL or about 24 mg/mL and water.

A hydrogel or a hydrogel composition of the present disclosure mayinclude a cellular component, for example, components of human adiposetissue, for example, adipose-derived stem cells, stromal vascularfraction cells, etc.

When injected or implanted in vivo, a hydrogel or a hydrogel compositionmay promote cell and/or tissue growth, including growth into the implantmaterial. For example, a hydrogel or hydrogel composition may stimulateangiogenesis, neovascularization, adipogenesis, collagenesis, cellinfiltration, tissue integration, and the like in vivo. In someembodiments, a hydrogel or a hydrogel composition may promote this typeof growth or activity to a greater extent than a hydrogel compositioncomprising hyaluronic acid having a weight concentration that is similarto the weight concentration of the crosslinked macromolecular matrixused in a hydrogel described herein. In some embodiments, a hydrogel ora hydrogel composition may promote this type of growth or activity to agreater extent than a hydrogel composition comprising water andhyaluronic acid at a concentration of about 24 mg/mL or about 16 mg/mL.In some embodiments, a hydrogel or a hydrogel composition may promotethis type of growth or activity to a greater extent than a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the silk fibroin component are not crosslinked

Once injected or implanted into a soft tissue, a hydrogel compositiondisclosed herein may stimulate angiogenesis, neovascularization,adipogenesis, and/or collagenesis. In an embodiment, a hydrogelcomposition disclosed herein stimulates angiogenesis,neovascularization, adipogenesis, and/or collagenesis to a greaterextent as compared to a hydrogel composition that is substantiallyidentical except that the hyaluronic acid component and the silk fibroincomponent are not crosslinked. In some embodiments, a hydrogelcomposition disclosed herein angiogenesis, neovascularization,adipogenesis, and/or collagenesis by at least about 50% at least about100%, at least about 150%, at least about 200%, at least 250%, at leastabout 300%, at least about 350%, at least about 400%, at least about450%, at least about 500%, at least about 750%, or at least about 1000%as compared to a hydrogel composition that is substantially identicalexcept that the hyaluronic acid component and the silk fibroin componentare not crosslinked. In some embodiments, a hydrogel compositiondisclosed herein angiogenesis, neovascularization, adipogenesis, and/orcollagenesis by about 50% to about 250%, about 50% to about 500%, about50% to about 1000%, about 100% to about 300%, about 100% to about 500%,about 100% to about 1000%, about 150% to about 400%, about 150% to about600%, about 150% to about 1000%, about 200% to about 500%, about 200% toabout 700%, or about 200% to about 1000% as compared to a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the silk fibroin component are not crosslinked. Insome embodiments, a hydrogel composition that is substantially identicalto a hydrogel composition disclosed herein except that the hyaluronicacid component and the silk fibroin component are not crosslinkedcomprises hyaluronic acid at a concentration of about 16 mg/mL or about24 mg/mL and water.

In another embodiment, a hydrogel composition disclosed hereinstimulates angiogenesis, neovascularization, adipogenesis, and/orcollagenesis to a greater extent as compared to adipose tissue with ahydrogel composition that is substantially identical except that thesilk fibroin component is absent. In some embodiments, a hydrogelcomposition disclosed herein angiogenesis, neovascularization,adipogenesis, and/or collagenesis by at least about 50% at least about100%, at least about 150%, at least about 200%, at least 250%, at leastabout 300%, at least about 350%, at least about 400%, at least about450%, at least about 500%, at least about 750%, or at least about 1000%as compared to a hydrogel composition that is substantially identicalexcept that the silk fibroin component is absent. In some embodiments, ahydrogel composition disclosed herein angiogenesis, neovascularization,adipogenesis, and/or collagenesis by about 50% to about 250%, about 50%to about 500%, about 50% to about 1000%, about 100% to about 300%, about100% to about 500%, about 100% to about 1000%, about 150% to about 400%,about 150% to about 600%, about 150% to about 1000%, about 200% to about500%, about 200% to about 700%, or about 200% to about 1000% as comparedto a hydrogel composition that is substantially identical except thatthe silk fibroin component is absent. In some embodiments, a hydrogelcomposition that is substantially identical to a hydrogel compositiondisclosed herein except that the silk fibroin component is absentcomprises hyaluronic acid at a concentration of about 16 mg/mL or about24 mg/mL and water.

Once injected or implanted into a soft tissue, a hydrogel compositiondisclosed herein may show infiltration and/or tissue integration ofcells from the soft tissue. In an embodiment, a hydrogel compositiondisclosed herein shows cell infiltration and/or tissue integration fromthe soft tissue to a greater extent as compared to a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the silk fibroin component are not crosslinked. Insome embodiments, a hydrogel composition disclosed herein shows enhancedcell infiltration and/or tissue integration by at least about 5% atleast about 10%, at least about 15%, at least about 20%, at least 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, or at least about 50% as compared to a hydrogel compositionthat is substantially identical except that the hyaluronic acidcomponent and the silk fibroin component are not crosslinked. In someembodiments, a hydrogel composition disclosed herein shows enhanced cellinfiltration and/or tissue integration by about 5% to about 25%, about5% to about 50%, about 10% to about 30%, about 10% to about 50%, about15% to about 40%, about 15% to about 50%, or about 20% to about 50% ascompared to a hydrogel composition that is substantially identicalexcept that the hyaluronic acid component and the silk fibroin componentare not crosslinked. In some embodiments, a hydrogel composition that issubstantially identical to a hydrogel composition disclosed hereinexcept that the hyaluronic acid component and the silk fibroin componentare not crosslinked comprises hyaluronic acid at a concentration ofabout 16 mg/mL or about 24 mg/mL and water.

In another embodiment, a hydrogel composition disclosed herein may showcell infiltration and/or tissue integration from the soft tissue to agreater extent as compared to a hydrogel composition that issubstantially identical except that the silk fibroin component isabsent. In some embodiments, a hydrogel composition disclosed hereinshows enhanced cell infiltration and/or tissue integration by at leastabout 5% at least about 10%, at least about 15%, at least about 20%, atleast 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, or at least about 50% as compared to a hydrogelcomposition that is substantially identical except that the silk fibroincomponent is absent. In some embodiments, a hydrogel compositiondisclosed herein shows enhanced cell infiltration and/or tissueintegration by about 5% to about 25%, about 5% to about 50%, about 10%to about 30%, about 10% to about 50%, about 15% to about 40%, about 15%to about 50%, or about 20% to about 50% as compared to a hydrogelcomposition that is substantially identical except that the silk fibroincomponent is absent. In some embodiments, a hydrogel composition that issubstantially identical to a hydrogel composition disclosed hereinexcept that the silk fibroin component is absent comprises hyaluronicacid at a concentration of about 16 mg/mL or about 24 mg/mL and water.

In some methods, hydrogel or a hydrogel composition may be mixed withtissue, for example, adipose tissue or fat tissue from the human being,such as human lipoaspirate, or from fat from another human being or ananimal. The tissue may comprise adipose-derived progenitor cells, forexample, adipose-derived stem cells. In some embodiments, methods areprovided for soft tissue augmentations and fat grafting using such celland filler compositions, which include autologous cells, for example,autologous, adipose-derived adult stem and/or progenitor cells. Theratio of hydrogel to fat in such a mixture may vary to provide thedesired results. The fat:hydrogel ratio is the weight of the fat dividedby the weight of hydrogel. For example, if 1 gram of fat is mixed with10 grams of hydrogel, the fat:hydrogel weight ratio is 0.1. In someembodiments, the fat tissue and the hydrogel may have a fat:hydrogelweight ratio of about 0.1 up to about 10. All other fat:hydrogel weightratios falling within this range are also contemplated and considered tobe within the scope of some embodiments. For example, the weight ratiomay be about 0.5 up to about 7, for example, about 1 up to about 5. Insome embodiments, the fat:hydrogel weight ratio is about 1 to about 3,for example, about 1, about 2, or about 3.

A combination or mixture of human fat tissue and hydrogel compositionmay then be injected or implanted into soft tissue of a human being, foraugmenting the breast for example. This may help to improve the survivaltime of grafted fat in autologous and other fat transfer procedures. Itmay also help to improve volume retention, reduce the variability inretained fat graft volume, and/or reduce inflammation as compared toinjecting fat tissue alone.

A hydrogel composition disclosed herein may show improved volumeretention after injection or implantation into a soft tissue. In anembodiment, a hydrogel composition disclosed herein shows improvedvolume retention after injection or implantation into a soft tissue ascompared to a hydrogel composition that is substantially identicalexcept that the hyaluronic acid component and the silk fibroin componentare not crosslinked. In some embodiments, a hydrogel compositiondisclosed herein shows improved volume retention after injection orimplantation into a soft tissue by at least about 5% at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,or at least about 50% as compared to a hydrogel composition that issubstantially identical except that the hyaluronic acid component andthe silk fibroin component are not crosslinked. In some embodiments, ahydrogel composition disclosed herein shows improved volume retentionafter injection or implantation into a soft tissue by about 5% to about25%, about 5% to about 50%, about 10% to about 30%, about 10% to about50%, about 15% to about 40%, about 15% to about 50%, or about 20% toabout 50% as compared to a hydrogel composition that is substantiallyidentical except that the hyaluronic acid component and the silk fibroincomponent are not crosslinked. In some embodiments, a hydrogelcomposition that is substantially identical to a hydrogel compositiondisclosed herein except that the hyaluronic acid component and the silkfibroin component are not crosslinked comprises hyaluronic acid at aconcentration of about 16 mg/mL or about 24 mg/mL and water.

In another embodiment, a hydrogel composition disclosed herein showsimproved volume retention after injection or implantation into a softtissue as compared to a hydrogel composition that is substantiallyidentical except that the silk fibroin component is absent. In someembodiments, a hydrogel composition disclosed herein shows improvedvolume retention after injection or implantation into a soft tissue byat least about 5% at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, or at least about 50% as comparedto a hydrogel composition that is substantially identical except thatthe silk fibroin component is absent. In some embodiments, a hydrogelcomposition disclosed herein shows improved volume retention afterinjection or implantation into a soft tissue by about 5% to about 25%,about 5% to about 50%, about 10% to about 30%, about 10% to about 50%,about 15% to about 40%, about 15% to about 50%, or about 20% to about50% as compared to a hydrogel composition that is substantiallyidentical except that the silk fibroin component is absent. In someembodiments, a hydrogel composition that is substantially identical to ahydrogel composition disclosed herein except that the silk fibroincomponent is absent comprises hyaluronic acid at a concentration ofabout 16 mg/mL or about 24 mg/mL and water.

A hydrogel composition disclosed herein may show decreased variabilityin volume retention after injection or implantation into a soft tissue.In an embodiment, a hydrogel composition disclosed herein showsdecreased variability in volume retention after injection orimplantation into a soft tissue as compared to a hydrogel compositionthat is substantially identical except that the hyaluronic acidcomponent and the silk fibroin component are not crosslinked. In someembodiments, a hydrogel composition disclosed herein shows decreasedvariability in volume retention after injection or implantation into asoft tissue by at least about 5% at least about 10%, at least about 15%,at least about 20%, at least 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, or at least about 50% ascompared to a hydrogel composition that is substantially identicalexcept that the hyaluronic acid component and the silk fibroin componentare not crosslinked. In some embodiments, a hydrogel compositiondisclosed herein shows decreased variability in volume retention afterinjection or implantation into a soft tissue by about 5% to about 25%,about 5% to about 50%, about 10% to about 30%, about 10% to about 50%,about 15% to about 40%, about 15% to about 50%, or about 20% to about50% as compared to a hydrogel composition that is substantiallyidentical except that the hyaluronic acid component and the silk fibroincomponent are not crosslinked. In some embodiments, a hydrogelcomposition that is substantially identical to a hydrogel compositiondisclosed herein except that the hyaluronic acid component and the silkfibroin component are not crosslinked comprises hyaluronic acid at aconcentration of about 16 mg/mL or about 24 mg/mL and water.

In another embodiment, a hydrogel composition disclosed herein showsdecreased variability in volume retention after injection orimplantation into a soft tissue as compared to a hydrogel compositionthat is substantially identical except that the silk fibroin componentis absent. In some embodiments, a hydrogel composition disclosed hereinshows decreased variability in volume retention after injection orimplantation into a soft tissue by at least about 5% at least about 10%,at least about 15%, at least about 20%, at least 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, or atleast about 50% as compared to a hydrogel composition that issubstantially identical except that the silk fibroin component isabsent. In some embodiments, a hydrogel composition disclosed hereinshows decreased variability in volume retention after injection orimplantation into a soft tissue by about 5% to about 25%, about 5% toabout 50%, about 10% to about 30%, about 10% to about 50%, about 15% toabout 40%, about 15% to about 50%, or about 20% to about 50% as comparedto a hydrogel composition that is substantially identical except thatthe silk fibroin component is absent. In some embodiments, a hydrogelcomposition that is substantially identical to a hydrogel compositiondisclosed herein except that the silk fibroin component is absentcomprises hyaluronic acid at a concentration of about 16 mg/mL or about24 mg/mL and water.

Once injected or implanted into a soft tissue, a hydrogel compositiondisclosed herein may reduce inflammation of the soft tissue. In anembodiment, a hydrogel composition disclosed herein reduces inflammationof the soft tissue as compared to a hydrogel composition that issubstantially identical except that the hyaluronic acid component andthe silk fibroin component are not crosslinked. In some embodiments, ahydrogel composition disclosed herein reduces inflammation of the softtissue by at least about 5% at least about 10%, at least about 15%, atleast about 20%, at least 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, or at least about 50% ascompared to a hydrogel composition that is substantially identicalexcept that the hyaluronic acid component and the silk fibroin componentare not crosslinked. In some embodiments, a hydrogel compositiondisclosed herein reduces inflammation of the soft tissue by about 5% toabout 25%, about 5% to about 50%, about 10% to about 30%, about 10% toabout 50%, about 15% to about 40%, about 15% to about 50%, or about 20%to about 50% as compared to a hydrogel composition that is substantiallyidentical except that the hyaluronic acid component and the silk fibroincomponent are not crosslinked. In some embodiments, a hydrogelcomposition that is substantially identical to a hydrogel compositiondisclosed herein except that the hyaluronic acid component and the silkfibroin component are not crosslinked comprises hyaluronic acid at aconcentration of about 16 mg/mL or about 24 mg/mL and water.

In another embodiment, a hydrogel composition disclosed herein reducesinflammation of the soft tissue as compared to a hydrogel compositionthat is substantially identical except that the silk fibroin componentis absent. In some embodiments, a hydrogel composition disclosed hereinreduces inflammation of the soft tissue by at least about 5% at leastabout 10%, at least about 15%, at least about 20%, at least 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, or at least about 50% as compared to a hydrogel composition that issubstantially identical except that the silk fibroin component isabsent. In some embodiments, a hydrogel composition disclosed hereinreduces inflammation of the soft tissue by about 5% to about 25%, about5% to about 50%, about 10% to about 30%, about 10% to about 50%, about15% to about 40%, about 15% to about 50%, or about 20% to about 50% ascompared to a hydrogel composition that is substantially identicalexcept that the silk fibroin component is absent. In some embodiments, ahydrogel composition that is substantially identical to a hydrogelcomposition disclosed herein except that the silk fibroin component isabsent comprises hyaluronic acid at a concentration of about 16 mg/mL orabout 24 mg/mL and water.

A hydrogel or a hydrogel composition may have improved physicalproperties that may help to encourage cell survival or proliferation. Insome embodiments, a hydrogel or a hydrogel composition may allowdiffusion of adipose tissue-specific growth factors or pro-angiogenicgrowth factors to a greater extent than a hydrogel compositioncomprising hyaluronic acid at a concentration of about 24 mg/mL or about16 mg/mL and water.

A hydrogel composition disclosed herein may show improved diffusion ofadipose tissue-specific growth factors or pro-angiogenic growth factors.In an embodiment, a hydrogel composition disclosed herein showsdiffusion of adipose tissue-specific growth factors or pro-angiogenicgrowth factors to a greater extent as compared to a hydrogel compositionthat is substantially identical except that the hyaluronic acidcomponent and the silk fibroin component are not crosslinked. In someembodiments, a hydrogel composition disclosed herein shows improveddiffusion of adipose tissue-specific growth factors or pro-angiogenicgrowth factors by at least about 25% at least about 50%, at least about75%, at least about 100%, at least 125%, at least about 150%, at leastabout 175%, at least about 200%, at least about 225%, or at least about250% as compared to a hydrogel composition that is substantiallyidentical except that the hyaluronic acid component and the silk fibroincomponent are not crosslinked. In some embodiments, a hydrogelcomposition disclosed herein shows improved diffusion of adiposetissue-specific growth factors or pro-angiogenic growth factors by about25% to about 100%, about 25% to about 150%, about 25% to about 250%,about 50% to about 100%, about 50% to about 150%, or about 50% to about250% as compared to a hydrogel composition that is substantiallyidentical except that the hyaluronic acid component and the silk fibroincomponent are not crosslinked. In some embodiments, a hydrogelcomposition that is substantially identical to a hydrogel compositiondisclosed herein except that the hyaluronic acid component and the silkfibroin component are not crosslinked comprises hyaluronic acid at aconcentration of about 16 mg/mL or about 24 mg/mL and water.

In another embodiment, a hydrogel composition disclosed herein showsdiffusion of adipose tissue-specific growth factors or pro-angiogenicgrowth factors to a greater extent as compared to a hydrogel compositionthat is substantially identical except that the silk fibroin componentis absent. In some embodiments, a hydrogel composition disclosed hereinshows improved diffusion of adipose tissue-specific growth factors orpro-angiogenic growth factors by at least about 25% at least about 50%,at least about 75%, at least about 100%, at least 125%, at least about150%, at least about 175%, at least about 200%, at least about 225%, orat least about 250% as compared to a hydrogel composition that issubstantially identical except that the silk fibroin component isabsent. In some embodiments, a hydrogel composition disclosed hereinshows improved diffusion of adipose tissue-specific growth factors orpro-angiogenic growth factors by about 25% to about 100%, about 25% toabout 150%, about 25% to about 250%, about 50% to about 100%, about 50%to about 150%, or about 50% to about 250% as compared to a hydrogelcomposition that is substantially identical except that the silk fibroincomponent is absent. In some embodiments, a hydrogel composition that issubstantially identical to a hydrogel composition disclosed hereinexcept that the silk fibroin component is absent comprises hyaluronicacid at a concentration of about 16 mg/mL or about 24 mg/mL and water.

A hydrogel or a hydrogel composition may be used to prepare a space inhuman or animal tissue. This may be done by injecting a hydrogel or ahydrogel composition into the tissue. After being injected, a hydrogelor a hydrogel composition may degrade over time, such as over a periodof about 1 week to about 3 months or about 2 weeks to about 6 weeks, tothereby create the space in the tissue. This may create a fertilenutrient bed through stimulated angiogenesis, cellular ingrowth,secretion of tropic factors, as well as creating space. An anestheticmay also be injected into the tissue, such as before injection of ahydrogel or hydrogel composition, or as part of a hydrogel composition.This may help reduce the pain of injection and allow the procedure to bedone as an outpatient procedure.

Once a hydrogel has degraded sufficiently to create a desired space, ahuman or animal fat composition may be injected into the space in thetissue. A fertile nutrient bed created as described above may help toimprove overall fat graft retention as compared to injecting fat withoutpreparing a space as described above.

Some embodiments include a packaged product comprising a device forfacilitating introduction, for example, a syringe loaded with a hydrogeland a needle. A syringe may be fitted with a needle of any size that isappropriate for injecting the hydrogel into the soft tissue of interest,such as a needle with about a #25, about a #30, or a larger gauge.

A filler comprising a hydrogel may be suitable for injection if it canbe injected into the soft tissue of interest without unreasonabledifficulty, and includes fillers that can be dispensed from syringeshaving gauge as low as about #30 or about #25 under normal manualpressure with a smooth extrusion plateau.

Injection of a hydrogel may provide a soft tissue augmentation thatmimics the natural components of the skin. A hydrogel may be injectedintradermally or subcutaneously to augment soft tissue and to repair orcorrect congenital anomalies, acquired defects, or cosmetic defects.Examples of such conditions include congenital anomalies such ashemifacial microsomia, malar and zygomatic hypoplasia, unilateralmammary hypoplasia, pectus excavatum, pectoralis agenesis (Poland'sanomaly), and velopharyngeal incompetence secondary to cleft palaterepair or submucous cleft palate (as a retropharyngeal implant);acquired defects (post traumatic, post surgical, or post infectious)such as depressed scars, subcutaneous atrophy (e.g., secondary todiscoid lupis erythematosis), keratotic lesions, enopthalmos in theunucleated eye (also superior sulcus syndrome), acne pitting of theface, linear scleroderma with subcutaneous atrophy, saddle-nosedeformity, Romberg's disease, and unilateral vocal cord paralysis; andcosmetic defects such as glabellar frown lines, deep nasolabial creases,circum-oral geographical wrinkles, sunken cheeks, and mammaryhypoplasia.

A hydrogel may comprise water and a crosslinked macromolecular matrix.Typically, a crosslinked molecular matrix may comprise a hyaluronic acidcomponent and a silk fibroin component, wherein the hyaluronic acidcomponent is crosslinked to the silk fibroin component by a crosslinkingcomponent. A crosslinking component may comprise a plurality ofcrosslink units, wherein at least a portion of the crosslink unitscomprise an ester bond or an amide bond.

A hydrogel or a hydrogel composition may be at least about 70%, about93%, or about 96% water by weight, and may approach 100% water byweight. A crosslinked macromolecular matrix may be about 0.01% to about30%, about 0.1% to about 7%, or about 0.2% to about 4% of the weight ofa hydrogel or a hydrogel composition. A hyaluronic acid component may beabout 0.005% to about 20%, about 0.1% to about 5% or about 0.2% to about2.5% of the total weight of a hydrogel or a hydrogel composition. A silkfibroin component may be about 0.01% to about 10%, about 0.03% to about2%, or about 0.05% to about 1.2% of the total weight of a hydrogel or ahydrogel composition.

A crosslinked macromolecular matrix for a hydrogel may be synthesized bycoupling a hyaluronic acid with a silk fibroin using a coupling agent,such as a carbodiimide. In these hydrogels, hyaluronic acid may serve asa biocompatible water-binding component, providing bulk andisovolumetric degradation. Additionally, silk fibroin may impart celladhesion and signaling domains to promote cell attachment, migration,and other cell functions such as extra-cellular matrix deposition. Thebiopolymers form homogeneous hydrogels with tunable composition,swelling, and mechanical properties. Compositions can be made to beinjectable for minimally invasive implantation through syringe andneedle.

Hyaluronic acid is a non-sulfated glycosaminoglycan that enhances waterretention and resists hydrostatic stresses. It is non-immunogenic andcan be chemically modified in numerous fashions. Hyaluronic acid may beanionic at pH ranges around or above the pKa of its carboxylic acidgroups. Unless clearly indicated otherwise, reference to hyaluronic acidherein may include its fully protonated, or nonionic form as depictedbelow, as well as any anionic forms and salts of hyaluronic acid, suchas sodium salts, potassium salts, lithium salts, magnesium salts,calcium salts, etc.

Hyaluronic Acid

Under certain conditions, a hyaluronic acid and a silk fibroin may becombined in an aqueous liquid in which both components are soluble. Ahyaluronic acid and a silk fibroin may then be crosslinked while bothare dissolved in an aqueous solution to form a hydrogel. Reactionconditions such as the concentration of hyaluronic acid, theconcentration of silk fibroin, the pH of the solution, and saltconcentration may be adjusted to help to prevent polyionic complexformation between anionic hyaluronic acid and cationic silk fibroin.They may also help to prevent silk fibroin microfibril formation, whichresults in precipitation from solution and may prevent crosslinking.

Some embodiments include a method of crosslinking hyaluronic acid andsilk fibroin. This method generally comprises a dissolution step, whichresults in an aqueous pre-reaction solution. In a dissolution step,hyaluronic acid and silk fibroin are dissolved in an aqueous solutionthat has a low pH and/or a salt to form an aqueous pre-reactionsolution.

A hyaluronic acid-silk fibroin crosslinking method further comprises anactivation step. In an activation step, an aqueous pre-reaction solutionis modified by at least adding a water soluble coupling agent and/or byincreasing the pH of the solution. If needed, a salt may also be addedto keep the hyaluronic acid and silk fibroin in solution at the higherpH. Thus, a crosslinking reaction mixture comprises hyaluronic acid andsilk fibroin dissolved or dispersed in an aqueous medium, a watersoluble coupling agent, and a salt, and has a higher pH than the aqueouspre-reaction solution from which it was derived. The crosslinkingreaction mixture is allowed to react to thereby crosslink the hyaluronicacid and the collagen.

In some embodiments, the pH of the aqueous pre-reaction solution may beincreased and a substantial amount of fiber formation may be allowed tooccur in the solution before adding the water soluble coupling agent. Insome embodiments, the water soluble coupling agent may be added to theaqueous pre-reaction solution before substantially any fiber formationoccurs.

A crosslinking reaction mixture can react to form a crosslinkedmacromolecular matrix. Since reaction occurs in an aqueous solution, acrosslinked macromolecular matrix may be dispersed in an aqueous liquidin hydrogel form as it is formed by a crosslinking reaction. Acrosslinked macromolecular matrix may be kept in hydrogel form because,in many instances, a crosslinked macromolecular matrix may be used inhydrogel form.

In some embodiments, an aqueous pre-reaction solution or a crosslinkingreaction mixture may further comprise about 10% to about 90% of anorganic solvent in which hyaluronic acid has poor solubility, such asethanol, methanol, isopropanol, or the like.

After a crosslinking reaction has occurred, the crosslinkedmacromolecular matrix may be particulated or homogenized through a mesh.This may help to form an injectable slurry or hydrogel. A mesh used forparticulating a crosslinked macromolecular matrix may have any suitablepore size depending upon the size of particles desired. In someembodiments, the mesh may have a pore size of about 10 microns to about100 microns, about 50 microns to about 70 microns, or about 60 microns.

A hydrogel comprising a crosslinked molecular matrix may be treated bydialysis for sterilization or other purposes. Dialysis may be carriedout by placing a semipermeable membrane between the hydrogel and anotherliquid so as to allow the hydrogel and the liquid to exchange moleculesor salts that can pass through the membrane.

A dialysis membrane may have a molecular weight cutoff that may vary.For example, the cutoff may be about 5,000 daltons to about 100,000daltons, about 10,000 daltons to about 30,000 daltons, or about 20,000daltons.

The dialysis may be carried out against a buffer solution, meaning thatthe liquid on the other side of the membrane from the hydrogel may be abuffer solution. In some embodiments, the buffer solution may be asterile phosphate buffer solution that may comprise phosphate buffer,potassium chloride, and/or sodium chloride. A sterile phosphate buffersolution may be substantially isosmotic with respect to humanphysiological fluid. Thus, when dialysis is complete, the liquidcomponent of a hydrogel may be substantially isosmotic with respect tohuman physiological fluid.

In some embodiments, a crosslinked macromolecular complex may furthercomprise an aqueous liquid. For example, the crosslinked macromolecularcomplex may absorb the aqueous liquid so that a hydrogel is formed. Anaqueous liquid may comprise water with a salt dissolved in it, such as aphosphate buffer, sodium chloride, potassium chloride, etc. In someembodiments, an aqueous liquid may comprise water, sodium chloride at aconcentration of about 100 mM to about 200 mM, potassium chloride at aconcentration of about 2 mM to about 3 mM, and phosphate buffer at aconcentration of about 5 mM to about 15 mM, wherein the pH of the liquidis about 7 to about 8.

In some embodiments, an anesthetic may be included in any compositioncomprising a crosslinked macromlecular complex in an amount effective tomitigate pain experienced upon injection of the composition. Examples ofan anesthetic may include, but are not limited to, ambucaine, amolanone,amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine,bupivacaine, butacaine, butamben, butanilicaine, butethamine,butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine,cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodon,dycyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine,beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine,hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate,levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methylchloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine,parethoxycaine, phenacaine, phenol, piperocaine, piridocaine,polidocanol, pramoxine, prilocaine, procaine, propanocaine,proparacaine, propipocaine, propoxycaine, psuedococaine, pyrrocaine,ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine,zolamine, and salts thereof. In some embodimenst, the at least oneanesthetic agent is lidocaine, such as in the form of lidocaine HCl. Theconcentration of lidocaine may vary. For example, some compositions mayhave about 0.1% to about 5%, about 0.2% to about 1.0%, or about 0.3%lidocaine by weight (w/w %) of the composition. The concentration oflidocaine in the compositions described herein can be therapeuticallyeffective meaning the concentration may be adequate to provide atherapeutic benefit without inflicting harm to the patient.

A hydrogel may be used in a soft tissue aesthetic product. An aestheticproduct includes any product that improves any aesthetic property of anypart of an animal or human being. A soft tissue aesthetic product maycomprise: an aesthetic device having a form suitable for injecting orimplanting into human tissue; and a label comprising instructions toinject or implant the aesthetic component into human tissue; wherein theaesthetic device comprises a crosslinked macromolecular matrix describedherein. Some products may comprise the crosslinked macromolecular matrixin hydrogel form.

Some embodiments include a method of improving an aesthetic quality ofan anatomic feature of a human being. Improving an aesthetic quality ofan anatomic feature of a human being includes improving any kind ofaesthetic quality including appearance, tactile sensation, etc., andimproving any anatomical feature, including those of the face, limbs,breasts, buttocks, hands, etc. Such a method may comprise injecting orimplanting an aesthetic device into a tissue of the human being tothereby improve the aesthetic quality of the anatomic feature; whereinthe aesthetic device comprises a crosslinked macromolecular matrixcomposition described herein. In some embodiments, the crosslinkedmacromolecular matrix used in the product may be in hydrogel form.

In some embodiments, a hydrogel of a crosslinked macromolecular complexmay have a storage modulus of about 1 Pa to about 10,000 Pa, about 50 Pato 10,000 Pa, about 50 Pa to about 6000 Pa, about 80 Pa to about 2000Pa, about 500 Pa to about 1000 Pa, about 500 Pa to about 4000 Pa, about500 Pa to about 5000 Pa, about 556 Pa, about 560 Pa, about 850 Pa, about852 Pa, about 1000 Pa, or any value in a range bounded by, or between,any of these values.

In some embodiments, a hydrogel of a crosslinked macromolecular complexmay have a loss modulus of about 1 Pa to about 500 Pa, about 10 Pa to200 Pa, about 100 Pa to about 200 Pa, about 20 Pa, about 131 Pa, about152 Pa, or any value in a range bounded by, or between, any of thesevalues.

In some embodiments, a hydrogel of a crosslinked macromolecular complexmay have an average extrusion force of about 10 N to about 50 N, about20 N to 30 N, or about 25 N, when the hydrogel is forced through a 30 Gneedle syringe by moving the plunger of a 1 mL syringe containing thehydrogel at a rate of 100 mm/min for about 11 mm, and measuring theaverage force from about 4 mm to about 10 mm.

A crosslinked macromolecular matrix may have tunable swelling propertiesbased on reaction conditions and hydrogel dilution. In some embodiments,a crosslinked macromolecular matrix may have a swelling ratio of about20 to about 200. A swelling ratio is the ratio of the weight of thecrosslinked macromolecular matrix after synthesis to the weight of thecrosslinked macromolecular matrix without any water. The crosslinkedmacromolecular matrix may have a swelling power of about 1 to about 7.The swelling power is the ratio of the weight of the crosslinkedmacromolecular matrix when it is saturated with water to the weight ofthe crosslinked macromolecular matrix after synthesis.

In a crosslinking reaction, the molecular weight of a hyaluronic acidmay vary. In some embodiments, a hyaluronic acid may have a molecularweight of about 200,000 daltons to about 10,000,000 daltons, about500,000 daltons to about 10,000,000 daltons, about 1,000,000 daltons toabout 5,000,000 daltons, or about 1,000,000 daltons to about 3,000,000daltons. When the crosslinking reaction occurs, the resultingcrosslinked macromolecular product may have a hyaluronic acid componentderived from the hyaluronic acid in the crosslinking reaction. Thus, theranges recited above may also apply to the molecular weight of ahyaluronic acid component, e.g., about 200,000 daltons to about10,000,000 daltons, about 500,000 daltons to about 10,000,000 daltons,about 1,000,000 daltons to about 5,000,000 daltons, or about 1,000,000daltons to about 3,000,000 daltons. The term “molecular weight” isapplied in this situation to a portion of the matrix even though thehyaluronic acid component may not actually be a separate molecule due tothe crosslinking. In some embodiments, a higher molecular weighthyaluronic acid may result in a crosslinked molecular matrix that mayhave a higher bulk modulus and/or less swelling

The concentration of hyaluronic acid in an aqueous pre-reaction solutionor a crosslinking reaction mixture may vary. In some embodiments,hyaluronic acid is present at about 3 mg/mL to about 100 mg/mL, about 6mg/mL to about 24 mg/mL, about 1 mg/mL to about 30 mg/mL, about 6 mg/mL,about 9 mg/L, about 12 mg/mL, about 15 mg/L, about 16 mg/mL, about 18mg/L, about 21 mg/L, or about 24 mg/mL. In some embodiments, higherhyaluronic acid concentration may lead to higher stiffness and/or moreswelling in the crosslinked macromolecular matrix.

Silk fibroin concentration in an aqueous pre-reaction solution or acrosslinking reaction mixture may vary. In some embodiments, silkfibroin may be present at a concentration of about 1 mg/mL to about 40mg/mL, about 1 mg/mL to about 15 mg/mL, about 3 mg/mL to about 12 mg/mL,about 1.7 mg/mL, about 3 mg/mL, about 6 mg/mL, about 8 mg/mL, or about12 mg/mL.

In some embodiments, the weight ratio of hyaluronic acid to silk fibroinin a aqueous pre-reaction solution or a aqueous pre-reaction solution ora crosslinking reaction mixture (e.g., [wt hyaluronic acid]/[wtcollagen]) may be about 0.5 to about 10, about 1 to about 7, about 0.5to about 3, about 1 to about 3, about 1 to about 2, about 1, about 2,about 3, about 3.5, about 4, about 5, 5.33, about 6, about 7, or anyweight ratio in a range bounded by, and/or between, any of these values.When the crosslinking reaction occurs, the resulting crosslinkedmacromolecular product may have a silk fibroin component derived fromthe silk fibroin in the crosslinking reaction. Thus, the resultingcrosslinked macromolecular matrix may have a weight ratio of hyaluronicacid component to silk fibroin component that corresponds to the weightratio in the crosslinking reaction, e.g., about 0.5 to about 10, about 1to about 7, about 0.5 to about 3, about 1 to about 3, about 1 to about2, about 1, about 2, about 3, about 3.5, about 4, about 5, 5.33, about6, about 7, or any weight ratio in a range bounded by, and/or between,any of these values. A higher weight ratio of hyaluronic acid to silkfibroin may result in a crosslinked macromolecular matrix with increasedswelling, decreased stiffness, and/or decreased cell adhesion.

Certain advantageous compositions of some embodiments includecompositions having a hyaluronic acid to silk fibroin weight ratio inthe range of about 25:1 to about 1:1. For example, the ratio can beabout 20:1, about 19:2, about 18:3, about 17:4, 3:3, about 12:6, about16:8, about 12:12, about 12:24, about 12:3, about 16:3, or about 20:3(mg/ml).

In some embodiments, the weight ratio of hyaluronic acid to silk fibroinin a aqueous pre-reaction solution or a aqueous pre-reaction solution ora crosslinking reaction mixture may be about 17 mg/mL of hyaluronic acidto about 4 mg/mL silk fibroin, about 20 mg/mL of hyaluronic acid toabout 1 mg/mL silk fibroin, or about 18 mg/mL of hyaluronic acid toabout 3 mg/mL silk fibroin.

An increase in the amount of both hyaluronic acid and silk fibroin mayresult in a crosslinked macromolecular matrix with increased stiffness.

A crosslinking reaction mixture may inlcude non-coordinating buffers.Any non-coordinating buffer may be used that is capable of buffering themixture and does not form coordinating complexes with coupling agents ormetal atoms. Examples of suitable non-coordinating buffers may include,but are not limited to, 2-(N-morpholino)ethanesulfonic acid (MES),3-(N-morpholino)propanesulfonic acid (MOPS),4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid (HEPES),3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPS),N-cyclohexyl-2-aminoethanesulfonic acid (CHES),N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), etc.

The concentration of a non-coordinating buffer may vary. For example,some aqueous pre-reaction solutions or crosslinking reaction mixturesmay have a buffer concentration in a range of about 10 mM to about 1 M,about 10 mM to about 500 mM, about 20 mM to about 100 mM, or about 25 mMto about 250 mM. Some aqueous pre-reaction solutions or crosslinkingreaction mixtures comprise MES at a concentration of about 20 mM toabout 200 mM, about 20 mM to about 100 mM, about 100 mM, or about 180mM.

Non-buffering salts may also be included in an aqueous pre-reactionsolution or a crosslinking reaction mixture as an alternative to, or inaddition, to buffering salts. Some examples may include sodium chloride,potassium chloride, lithium chloride, potassium bromide, sodium bromide,lithium bromide, and the like. The concentration of a non-buffering saltmay vary. For example, some mixtures may have a non-buffering saltconcentration in a range of about 10 mM to about 1 mM, about 30 mM toabout 500 mM, or about 50 mM to about 300 mM. In some embodiments,sodium chloride may be present at a concentration in a range of about0.5% w/v to about 2% about 0.9% w/v, about 1.6% w/v, about 20 mM toabout 1 mM, about 40 mM to about 500 mM, about 50 to 300 mM, about 80 mMto about 330 mM, about 150 mM, or about 270 mM.

The pH of an aqueous pre-reaction solution may be lower than the pH of acrosslinking reaction mixture. If the salt content of the aqueouspre-reaction solution is low, the pH may be lower to enhance solubilityof the hyaluronic acid and the silk fibroin. If the salt content ishigher, the pH may be higher in the aqueous pre-reaction solution. Insome embodiments, the pH of the aqueous pre-reaction mixture is about 1to about 8, about 3 to about 8, about 4 to about 6, about 4.7 to about7.4, or about 5.4. For low salt concentrations, the pH may be about 1 toabout 4 or about 1 to about 3. In some embodiments, a pH of around 5.4may result in a crosslinked macromolecular matrix having higherstiffness and/or lower swelling.

Any water-soluble coupling agent may be used that can crosslinkhyaluronic acid to silk fibroin. Some non-limiting examples of acoupling agent include carbodiimides such asN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC),or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), etc.Carbodiimide coupling agents may facilitate ester or amide bondformation without becoming part of the linkage. However, other couplingagents that become part of the crosslinking group may be used. Theconcentration of a coupling agent may vary. In some embodiments, acoupling agent may be present at about 2 mM to about 150 mM, about 2 mMto about 50 mM, about 20 mM to about 100 mM, or about 50 mM. In someembodiments, the coupling agent is EDC that is present at aconcentration of about 20 mM to about 100 mM, about 2 mM to about 50 mM,or about 50 mM.

An activating agent may be used to increase the rate of the crosslinkingreaction and the number of crosslink units in the final product. In someembodiments, an activating agent may be a triazole such ashydroxybenzotriazole (HOBT) or 1-hydroxy-7-azabenzotriazole (HOAT); afluorinated phenol such as pentafluorophenol; a succinimide such asN-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (sulfoNHS), andthe like.

The concentration of an activating agent may vary. In some embodiments,the activating agent may have a concentration of about 2 mM to about 200mM, about 2 mM to about 50 mM, about 20 mM to about 100 mM, or about 50mM. In some embodiments, the activating agent may be NHS or sulfoNHS isat a concentration of about 2 mM to about 50 mM. In some embodiments,the activating agent may be N-hydroxysulfosuccinimide, sodium salt, at aconcentration of about 20 mM to about 100 mM, or about 50 Mm.

Crosslinking HA via EDC chemistry involves the use of small multi aminecross linkers, which form amide bonds with the carboxylic functionalgroups of HA chains. In ideal condition, EDC activates the carboxylicacid groups of HA, and the activated carboxylic acid groups then reactwith the amines. Crosslinking is usually done at pH between 4-7 andtemperatures between 20 and 37° C., conditions at which degradation ofHA is minimal. Linear diamine cross linkers like hexamethylene diamine(HMDA), lysine, lysine methyl ester or lysine ethyl ester, have beenused to crosslink HA for various applications. Protein additives withhigh lysine content such as Collagen can also be used. Crosslinking HAvia EDC chemistry without the use of a multiamine cross linker resultsin the formation of ester bonds between carboxylic acid groups and thehydroxyl groups of HA. Ester bonds are very labile, and are easilyhydrolyzed at high temperatures. HA hydrogels made by ester crosslinking are generally not robust and cannot be sterilized with moiststeam.

The present disclosure includes a composition comprising a gel phaseincluding a hydrogel comprising a silk fibroin covalently attached to anHA (“the composition”).

The silk fibroin used for preparing the composition is an intermediatein the silk hydrogel production process and a direct precursor to thehydrogel material. The depolymerized silk fibroin can be made from rawcocoons, previously degummed silk or any other partially cleaned silk.This may also include material commonly termed as “waste” from thereeling process, i.e., short fragments of raw or degummed silk, the soleprecaution being that the silk must be substantially cleaned of sericinprior to making fibroin solution and inducing gel formation. Aparticular source of raw silk is from common domesticated silkworm B.mori, though several other sources of silk may be appropriate. Thisincludes other strains of Bombycidae including Antheraea pernyi,Antheraea yamamai, Antheraea mylitta, Antheraea assama, and Philosamiacynthia ricini, as well as silk producing members of the familiesSaturnidae, Thaumetopoeidae, and silk-producing members of the orderAraneae. The material may also be obtained from other spider,caterpillar, or recombinant sources.

A hydrogel disclosed herein provides for a depolymerized silk fibroinand/or silk fibroin that are substantially free of sericin. Methods forperforming sericin extraction have been described in pending U.S. patentapplication Ser. No. 10/008,924, U.S. Publication No. 2003/0100108,Matrix for the production of tissue engineered ligaments, tendons andother tissue. That application refers to cleaned fibroin fibers spuninto yarns, used to create a porous, elastic matrix suitable as asubstrate for applications requiring very high tensile strength, such asbioengineered ligaments and tendons.

Extractants such as urea solution, hot water, enzyme solutions includingpapain among others, which are known in the art to remove sericin fromfibroin would also be acceptable for generation of the silk. Mechanicalmethods may also be used for the removal of sericin from silk fibroin.This includes but is not limited to ultrasound, abrasive scrubbing andfluid flow. The rinse post-extraction is conducted preferably withvigorous agitation to remove substantially any ionic contaminants,soluble, and in soluble debris present on the silk as monitored throughmicroscopy and solution electrochemical measurements. A criterion isthat the extractant predictably and repeatably remove the sericin coatof the source silk without significantly compromising the molecularstructure of the fibroin. For example, an extraction may be evaluatedfor sericin removal via mass loss, amino acid content analysis, andscanning electron microscopy. Fibroin degradation may in turn bemonitored by FTIR analysis, standard protein gel electrophoresis andscanning electron microscopy.

In certain cases, the silk utilized for making the composition has beensubstantially depleted of its native sericin content (i.e., ≤4% (w/w)residual sericin in the final extracted silk). Alternatively, higherconcentrations of residual sericin may be left on the silk followingextraction or the extraction step may be omitted. In preferred someembodiments, the sericin-depleted silk fibroin has, e.g., about 0% toabout 4% (w/w) residual sericin. In the most preferred some embodiments,the sericin-depleted silk fibroin has, e.g., about 1% to 3% (w/w)residual sericin.

In certain cases, the silk utilized for generation of a silk hydrogel isentirely free of its native sericin content. As used herein, the term“entirely free (i.e., “consisting of” terminology) means that within thedetection range of the instrument or process being used, the substancecannot be detected or its presence cannot be confirmed.

The water soluble or dissolved silk can be prepared by a 4 hourdigestion at 60° C. of pure silk fibroin at a concentration of 200 g/Lin a 9.3 M aqueous solution of lithium bromide to a silk concentrationof 20% (w/v). This process may be conducted by other means provided thatthey deliver a similar degree of dissociation to that provided by a 4hour digestion at 60° C. of pure silk fibroin at a concentration of 200g/L in a 9.3 M aqueous solution of lithium bromide. The primary goal ofthis is to create uniformly and repeatably dissociated silk fibroinmolecules to ensure similar fibroin solution properties and,subsequently, device properties. Less substantially dissociated silksolution may have altered gelation kinetics resulting in differing finalgel properties. The degree of dissociation may be indicated byFourier-transform Infrared Spectroscopy (FTIR) or x-ray diffraction(XRD) and other modalities that quantitatively and qualitatively measureprotein structure. Additionally, one may confirm that heavy and lightchain domains of the silk fibroin dimer have remained intact followingsilk processing and dissolution. This may be achieved by methods such asstandard protein sodium-dodecyl-sulfate polyacrylamide gelelectrophoresis (SDS-PAGE), which assess molecular weight of theindependent silk fibroin domains.

System parameters which may be modified in the initial dissolution ofsilk include but are not limited to solvent type, silk concentration,temperature, pressure, and addition of mechanical disruptive forces.Solvent types other than aqueous lithium bromide may include but are notlimited to aqueous solutions, alcohol solutions,1,1,1,3,3,3-hexafluoro-2-propanol, and hexafluoroacetone,1-butyl-3-methylimidazolium. These solvents may be further enhanced byaddition of urea or ionic species including lithium bromide, calciumchloride, lithium thiocyanate, zinc chloride, magnesium salts, sodiumthiocyanate, and other lithium and calcium halides would be useful forsuch an application. These solvents may also be modified throughadjustment of pH either by addition of acidic of basic compounds.

Cross-linking can also be accomplished without exogenous cross-linkersby utilizing reactive groups on the molecules being conjugated. Methodsfor chemically cross-linking peptide molecules are generally known inthe art, and a number of hetero- and homobifunctional agents aredescribed in, e.g., U.S. Pat. Nos. 4,355,023, 4,657,853, 4,676,980,4,925,921, and 4,970,156, and Immuno Technology Catalogue and Handbook,Pierce Chemical Co. (1989), each of which is incorporated herein byreference. Such conjugation, including cross-linking, should beperformed so as not to substantially affect the desired function of thepeptide oligomer or entity conjugated thereto, including therapeuticagents, and moieties capable of binding substances of interest.

It will be apparent to one skilled in the art that alternative linkerscan be used to link peptides, for example the use of chemical proteincrosslinkers. For example homobifunctional crosslinker such asdisuccinimidyl-suberimidate-dihydrochloride;dimethyl-adipimidate-dihydrochloride; 1,5,-2,4dinitrobenezene orheterobifunctional crosslinkers such as N-hydroxysuccinimidyl2,3-dibromopropionate; 1ethyl-3-[3-dimethylaminopropyl] carbodiimidehydrochloride; andsuccinimidyl4-[n-maleimidomethyl]-cyclohexane-1-carboxylate.

A composition disclosed herein is typically biodegradable, bioerodible,and/or bioresorbable. In an embodiment, a silk fibroin cross linked to ahyaluronic acid hydrogel disclosed herein has a protein structure thatmakes the hydrogel resist biodegradation, bioerosion, and/orbioresorption. In some embodiments, a hydrogel is resistant tobiodegradation, bioerosion, and/or bioresorption for, e.g., betweenabout 10 days to about 180 days. In some embodiments, a hydrogel isresistant to biodegradation, bioerosion, and/or bioresorption for, e.g.,about 30 day to about 90 days. In some embodiments, a hydrogel isresistant to biodegradation, bioerosion, and/or bioresorption for, e.g.,about 20 days to 90 days.

In yet another embodiment, a silk fibroin hydrogel disclosed herein hasa protein structure that substantially includes β-turn and β-strandregions. In some embodiments, a hydrogel has a protein structureincluding, e.g., between about 10% to about 100% β-turn and β-strandregions. In some embodiments, a hydrogel has a protein structureincluding, e.g., between about 20% to about 70% β-turn and β-strandregions. In some embodiments, a hydrogel has a protein structureincluding, e.g., between about 30% to about 50% β-turn and β-strandregions.

In yet another embodiment, a silk fibroin hydrogel disclosed herein hasa protein structure that is substantially-free of α-helix and randomcoil regions. In some embodiments, a hydrogel has a protein structureincluding, e.g., between about 5% to about 50% α-helix and random coilregions. In some preferred some embodiments, a hydrogel has a proteinstructure including, e.g., between about 10% to about 40% α-helix andrandom coil regions. In the most preferred some embodiments, a hydrogelhas a protein structure including, e.g., between about 15% to about 35%α-helix and random coil regions.

Aspects of the present specification provide, in part, a silk fibroinhydrogel having hardness. Hardness refers to various properties of anobject in the solid phase that gives it high resistance to various kindsof shape change when force is applied. Hardness is measured using adurometer and is a unitless value that ranges from zero to 100. Theability or inability of a hydrogel to be easily compressed will affectits suitability for application in different tissue replacement roles,i.e., mechanical compliance as bone, fat, connective tissue. Hardnesswill also affect the ability of a hydrogel to be effectively comminuted,the reason being that a hard material may be more easily andconsistently comminuted. Hardness will also affect extrudability, as asoft material may be more readily able to be slightly compressed duringinjection to pack with other particles or change shape to pass through asyringe barrel or needle.

In an embodiment, a silk fibroin hydrogel exhibits low hardness. In someembodiments, a silk fibroin hydrogel exhibits a hardness of, e.g.,between about 5 to about 40. In some preferred some embodiments, a silkfibroin hydrogel exhibits a hardness of, e.g., between about 10 to about30. In the most preferred some embodiments, a silk fibroin hydrogelexhibits a hardness of, e.g., between about 15 to about 35.

In an embodiment, a silk fibroin hydrogel exhibits medium hardness. Insome embodiments, a silk fibroin hydrogel exhibits a hardness of, e.g.,about 40 to about 65. In some preferred some embodiments, a silk fibroinhydrogel exhibits a hardness of, e.g., about 30 to about 55. In the mostpreferred some embodiments, a silk fibroin hydrogel exhibits a hardnessof, e.g., about 45 to about 60.

In another embodiment, a silk fibroin hydrogel exhibits high hardness.In some embodiments, a silk hydrogel exhibits a hardness of, e.g.,between about 65 to about 95. In some preferred some embodiments, a silkhydrogel exhibits a hardness of, e.g., between about 70 to about 90. Inthe most preferred some embodiments, a silk hydrogel exhibits a hardnessof, e.g., between about 75 to about 85.

In an embodiment, a silk fibroin hydrogel exhibits high resistance todeformation. In some embodiments, a silk fibroin hydrogel exhibitsresistant to deformation of, e.g., about 100% to about 85%. In somepreferred some embodiments, a silk fibroin hydrogel exhibits resistantto deformation of, e.g., about 95% to about 80%. In the most preferredsome embodiments, a silk fibroin hydrogel exhibits resistant todeformation of, e.g., about 93% to about 78%.

A silk fibroin hydrogel exhibits an elastic modulus. Elastic modulus, ormodulus of elasticity, refers to the ability of a hydrogel material toresists deformation, or, conversely, an object's tendency to benon-permanently deformed when a force is applied to it. The elasticmodulus of an object is defined as the slope of its stress-strain curvein the elastic deformation region: λ=stress/strain, where λ is theelastic modulus in Pascal's; stress is the force causing the deformationdivided by the area to which the force is applied; and strain is theratio of the change caused by the stress to the original state of theobject. Specifying how stresses are to be measured, includingdirections, allows for many types of elastic moduli to be defined. Thethree primary elastic moduli are tensile modulus, shear modulus, andbulk modulus.

Tensile modulus (E) or Young's modulus is an objects response to linearstrain, or the tendency of an object to deform along an axis whenopposing forces are applied along that axis. It is defined as the ratioof tensile stress to tensile strain. It is often referred to simply asthe elastic modulus. The shear modulus or modulus of rigidity refers toan object's tendency to shear (the deformation of shape at constantvolume) when acted upon by opposing forces. It is defined as shearstress over shear strain. The shear modulus is part of the derivation ofviscosity. The shear modulus is concerned with the deformation of asolid when it experiences a force parallel to one of its surfaces whileits opposite face experiences an opposing force (such as friction). Thebulk modulus (K) describes volumetric elasticity or an object'sresistance to uniform compression, and is the tendency of an object todeform in all directions when uniformly loaded in all directions. It isdefined as volumetric stress over volumetric strain, and is the inverseof compressibility. The bulk modulus is an extension of Young's modulusto three dimensions.

In another embodiment, a silk fibroin hydrogel exhibits a tensile and/orshear modulus. In some embodiments, a silk fibroin hydrogel exhibits atensile modulus of, e.g., about 1 MPa to about 30 GPa. In some preferredsome embodiments, a silk fibroin hydrogel exhibits a tensile modulus of,e.g., about 5 MPa to about 25 GPa. In some embodiments, a silk fibroinhydrogel exhibits a tensile modulus of about 20 MPa to about 15 GPa.

In another embodiment, a silk fibroin hydrogel exhibits a bulk modulus.In some embodiments, a silk fibroin hydrogel exhibits a bulk modulus of,e.g., about 5 GPa to about 100 GPa. In some preferred some embodiments,a silk fibroin hydrogel exhibits a bulk modulus of, e.g., about 10 GPato about 90 GPa. In the most preferred some embodiments, a silk fibroinhydrogel exhibits a bulk modulus of, e.g., about 25 GPa to about 85 GPa.

A silk fibroin hydrogel exhibits high tensile strength. Tensile strengthhas three different definitional points of stress maxima. Yield strengthrefers to the stress at which material strain changes from elasticdeformation to plastic deformation, causing it to deform permanently.Ultimate strength refers to the maximum stress a material can withstandwhen subjected to tension, compression or shearing. It is the maximumstress on the stress-strain curve. Breaking strength refers to thestress coordinate on the stress-strain curve at the point of rupture, orwhen the material pulls apart.

In another embodiment, a silk fibroin hydrogel exhibits high yield, highultimate, and/or high breaking strength relative to other polymerclasses. In some embodiments, a silk fibroin hydrogel exhibits a yieldstrength of, e.g., about 0.1 MPa to about 500 MPa. In some preferredsome embodiments, a silk fibroin hydrogel exhibits a yield strength of,e.g., about 5 MPa to about 400 MPa. In the most preferred someembodiments, a silk fibroin hydrogel exhibits a yield strength of e.g.,about 20 MPa to about 300 MPa.

Aspects of the present specification provide, in part, a silk fibroinhydrogel having a transparency and/or translucency. Transparency (alsocalled pellucidity or diaphaneity) is the physical property of allowinglight to pass through a material, whereas translucency (also calledtranslucence or translucidity) only allows light to pass throughdiffusely. The opposite property is opacity. Transparent materials areclear, while translucent ones cannot be seen through clearly. The silkfibroin hydrogels disclosed herein may, or may not, exhibit opticalproperties such as transparency and translucency. In certain cases,e.g., superficial line filling, it would be an advantage to have anopaque hydrogel. In other cases such as development of a lens or a“humor” for filling the eye, it would be an advantage to have atranslucent hydrogel. These properties could be modified by affectingthe structural distribution of the hydrogel material. Factors used tocontrol a hydrogel's optical properties include, without limitation,silk fibroin concentration, gel crystallinity, and hydrogel homogeneity.

When light encounters a material, it can interact with it in severaldifferent ways. These interactions depend on the nature of the light(its wavelength, frequency, energy, etc.) and the nature of thematerial. Light waves interact with an object by some combination ofreflection, and transmittance with refraction. As a result, an opticallytransparent material allows much of the light that falls on it to betransmitted, with little light being reflected. Materials which do notallow the transmission of light are called optically opaque or simplyopaque.

In an embodiment, a silk fibroin hydrogel is optically transparent. Insome embodiments, a silk fibroin hydrogel transmits, e.g., between about75% to about 100% of the light. In some preferred some embodiments, asilk fibroin hydrogel transmits, e.g., between about 80% to about 90% ofthe light. In the most preferred some embodiments, a silk fibroinhydrogel transmits, e.g., between about 85% to about 90% of the light.

In another embodiment, a silk fibroin hydrogel is optically opaque. Insome embodiments, a silk fibroin hydrogel transmits, e.g., between about5% to about 75% of the light. In some preferred some embodiments, a silkfibroin hydrogel transmits, e.g., between about 10% to about 70% of thelight. In the most preferred some embodiments, a silk fibroin hydrogeltransmits, e.g., between about 15% to about 65% of the light.

In an embodiment, a silk fibroin hydrogel is optically translucent. Insome embodiments, a silk fibroin hydrogel diffusely transmits, e.g.,between about 75% to about 100% of the light. In some preferred someembodiments, a silk fibroin hydrogel diffusely transmits, e.g., betweenabout 80% to about 95% of the light. In some the most preferred someembodiments, a silk fibroin hydrogel diffusely transmits, e.g., betweenabout 85% to about 95% of the light.

After formation of a hydrogel described herein, the hydrogel can furtherprocessed. For example, to remove enhancer species and become a morecomplete, the formed hydrogel may be leeched against a solvent, such as,e.g., water, under ambient temperature and pressure conditions for threedays with five changes of water. The hydrogel may be leeched againstultra-pure water of a volume at least 100-times that of the gel. Morespecifically, for example, the gels may be placed in a bulk of purifiedwater and the rinse changed at hours 12, 24 and 48 with 15 mL gel per1.5 L water. The number of rinses and volume ratios involved may bealtered so long as the resultant hydrogel is substantially free ofresidual gelation enhancer.

A composition disclosed herein may be formulated using materialprocessing constraints such as silk concentration and salineconcentration to tailor material longevity in vivo. In one example, asilk hydrogel might be tailored for a persistence of five weeks to sixweeks in vivo by using a 1%-3% (w/v) silk gel with 25%-50% (v/v) salinecarrier. In another example, a silk hydrogel might be tailored for apersistence of two months to three months in vivo by using a 3%-5% (w/v)silk gel with 20%-40% (v/v) saline. In another example, a silk hydrogelmight be tailored for a persistence of 5-6 months by using 4-6% (w/v)silk gel with 20-40% (v/v) saline. In another example, a silk hydrogelmight be tailored for a persistence of 7-10 months by using a 6-8% (w/v)silk gel with 20-30% (v/v) saline. The persistence of these materialsmight also be increased or decreased by increasing or decreasingparticle size respectively.

Gel emulsion saline content and gel silk concentration could be used tomodify the mechanical profile of the silk gel materials for particularapplications. For example, a gel emulsion of about 1% (w/v) to about 5%(w/v) silk gel concentration with 5%-95% lubricant (e.g., 5%-95% (w/v)saline/PBS) may be useful as a dermal filler, bulking agent, camouflageagent, intramuscular or sub-Q filler, or pharmaceutical delivery vector.A gel emulsion of, for example, about 5% (w/v) to about 8% (w/v) silkgel concentration with 0% to about 30% lubricant fluid may be useful inbone defects or cartilage defects.

Aspects of the present specification provide, in part, a compositioncomprising a gel phase including a hydrogel comprising a matrix polymer.The compositions disclosed herein can further comprise a hydrogelcomprising one or more matrix polymers in addition to hydrogel particlescomprising silk fibroin, or a hydrogel comprising one or more matrixpolymers and silk fibroin. As used herein, the term “matrix polymer”refers to a polymer that can become part of and/or function as anextracellular matrix polymer and pharmaceutically acceptable saltsthereof. Non-limiting examples of a matrix polymer include aglycosaminoglycan like chondroitin sulfate, dermatan sulfate, keratansulfate, hyaluronan; a lubricin; a polysaccharide, and an elasticprotein (like silk protein, resilin, resilin-like polypeptides (RLPs),elastin (including tropoelastin, fibrillin and fibullin), elastin-likepolypeptides (ELPs), gluten (including gliadin and glutenin), abductin,byssus, and collagen). Non-limiting examples of a pharmaceuticallyacceptable salt of a matrix polymer includes sodium salts, potassiumsalts, magnesium salts, calcium salts, and combinations thereof. Matrixpolymers useful in the compositions and methods disclosed herein aredescribed in, e.g., Piron and Tholin, Polysaccharide Crosslinking,Hydrogel Preparation, Resulting Polysaccharides(s) and Hydrogel(s), usesThereof, U.S. Patent Publication 2003/0148995; Lebreton, Cross-Linkingof Low and High Molecular Weight Polysaccharides Preparation ofInjectable Monophase Hydrogels; Lebreton, Viscoelastic SolutionsContaining Sodium Hyaluronate and Hydroxypropyl Methyl Cellulose,Preparation and Uses, U.S. Patent Publication 2008/0089918; Lebreton,Hyaluronic Acid-Based Gels Including Lidocaine, U.S. Patent Publication2010/0028438; and Polysaccharides and Hydrogels thus Obtained, U.S.Patent Publication 2006/0194758; and Di Napoli, Composition and Methodfor Intradermal Soft Tissue Augmentation, International PatentPublication WO 2004/073759, each of which is hereby incorporated byreference in its entirety.

Aspects of the present specification provide, in part, a compositioncomprising a hyaluronan. As used herein, the term “hyaluronic acid” issynonymous with “HA”, “hyaluronic acid”, and “hyaluronate” refers to ananionic, non-sulfated glycosaminoglycan polymer comprising disaccharideunits, which themselves include D-glucuronic acid andD-N-acetylglucosamine monomers, linked together via alternating β-1,4and β-1,3 glycosidic bonds and pharmaceutically acceptable saltsthereof. Hyaluronan can be purified from animal and non-animal sources.Polymers of hyaluronan can range in size from about 5,000 Da to about20,000,000 Da. Any hyaluronan is useful in the compositions disclosedherein with the proviso that the hyaluronan improves a condition of theskin, such as, e.g., hydration or elasticity. Non-limiting examples ofpharmaceutically acceptable salts of hyaluronan include sodiumhyaluronan, potassium hyaluronan, magnesium hyaluronan, calciumhyaluronan, and combinations thereof.

Aspects of the present specification provide, in part, a compositioncomprising a crosslinked matrix polymer. As used herein, the term“crosslinked” refers to the intermolecular bonds joining the individualpolymer molecules, or monomer chains, into a more stable structure likea gel. As such, a crosslinked matrix polymer has at least oneintermolecular bond joining at least one individual polymer molecule toanother one. Matrix polymers disclosed herein may be crosslinked usingdialdehydes and disufides crosslinking agents including, withoutlimitation, multifunctional PEG-based cross linking agents, divinylsulfones, diglycidyl ethers, and bis-epoxides. Non-limiting examples ofhyaluronan crosslinking agents include divinyl sulfone (DVS),1,4-butanediol diglycidyl ether (BDDE),1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO),biscarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE),adipic dihydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS),hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane,or combinations thereof.

Aspects of the present specification provide, in part, a compositioncomprising a crosslinked matrix polymer having a degree of crosslinking.As used herein, the term “degree of crosslinking” refers to thepercentage of matrix polymer monomeric units that are bound to across-linking agent, such as, e.g., the disaccharide monomer units ofhyaluronan. Thus, a composition that that has a crosslinked matrixpolymer with a 4% degree of crosslinking means that on average there arefour crosslinking molecules for every 100 monomeric units. Every otherparameter being equal, the greater the degree of crosslinking, theharder the gel becomes. Non-limiting examples of a degree ofcrosslinking include about 1% to about 15%.

In an embodiment, a composition comprises an uncrosslinked hyaluronanwhere the uncrosslinked hyaluronan comprises a combination of both highmolecular weight hyaluronan and low molecular weight hyaluronan in aratio of about 20:1, about 15:1, about 10:1, about 5:1, about 1:1, about1:5 about 1:10, about 1:15, or about 1:20.

In another embodiment, a composition comprises an uncrosslinkedhyaluronan where the uncrosslinked hyaluronan comprises a combination ofboth high molecular weight hyaluronan and low molecular weighthyaluronan, in various ratios. As used herein, the term “high molecularweight hyaluronan” refers to a hyaluronan polymer that has a molecularweight of 1,000,000 Da or greater. Non-limiting examples of a highmolecular weight hyaluronan include a hyaluronan of about 1,500,000 Da,a hyaluronan of about 2,000,000 Da, a hyaluronan of about 2,500,000 Da,a hyaluronan of about 3,000,000 Da, a hyaluronan of about 3,500,000 Da,a hyaluronan of about 4,000,000 Da, a hyaluronan of about 4,500,000 Da,and a hyaluronan of about 5,000,000 Da. As used herein, the term “lowmolecular weight hyaluronan” refers to a hyaluronan polymer that has amolecular weight of less than 1,000,000 Da. Non-limiting examples of alow molecular weight hyaluronan include a hyaluronan of about 200,000Da, a hyaluronan of about 300,000 Da, a hyaluronan of about 400,000 Da,a hyaluronan of about 500,000 Da, a hyaluronan of about 600,000 Da, ahyaluronan of about 700,000 Da, a hyaluronan of about 800,000 Da, and ahyaluronan of about 900,000 Da.

In other some embodiments, a composition comprises a crosslinkedhyaluronan where the crosslinked hyaluronan has a mean molecular weightof, e.g., about 1,000,000 Da, about 1,500,000 Da, about 2,000,000 Da,about 2,500,000 Da, about 3,000,000 Da, about 3,500,000 Da, about4,000,000 Da, about 4,500,000 Da, or about 5,000,000 Da. In yet othersome embodiments, a composition comprises a crosslinked hyaluronan wherethe crosslinked hyaluronan has a mean molecular weight of, e.g., atleast 1,000,000 Da, at least 1,500,000 Da, at least 2,000,000 Da, atleast 2,500,000 Da, at least 3,000,000 Da, at least 3,500,000 Da, atleast 4,000,000 Da, at least 4,500,000 Da, or at least 5,000,000 Da. Instill other some embodiments, a composition comprises a crosslinkedhyaluronan where the crosslinked hyaluronan has a mean molecular weightof, e.g., about 1,000,000 Da to about 5,000,000 Da, about 1,500,000 Dato about 5,000,000 Da, about 2,000,000 Da to about 5,000,000 Da, about2,500,000 Da to about 5,000,000 Da, about 2,000,000 Da to about3,000,000 Da, about 2,500,000 Da to about 3,500,000 Da, or about2,000,000 Da to about 4,000,000 Da.

In other some embodiments, a composition comprises an uncrosslinkedhyaluronan where the uncrosslinked hyaluronan has a mean molecularweight of, e.g., about 1,000,000 Da, about 1,500,000 Da, about 2,000,000Da, about 2,500,000 Da, about 3,000,000 Da, about 3,500,000 Da, about4,000,000 Da, about 4,500,000 Da, or about 5,000,000 Da. In yet othersome embodiments, a composition comprises an uncrosslinked hyaluronanwhere the uncrosslinked hyaluronan has a mean molecular weight of, e.g.,at least 1,000,000 Da, at least 1,500,000 Da, at least 2,000,000 Da, atleast 2,500,000 Da, at least 3,000,000 Da, at least 3,500,000 Da, atleast 4,000,000 Da, at least 4,500,000 Da, or at least 5,000,000 Da. Instill other some embodiments, a composition comprises an uncrosslinkedhyaluronan where the uncrosslinked hyaluronan has a mean molecularweight of, e.g., about 1,000,000 Da to about 5,000,000 Da, about1,500,000 Da to about 5,000,000 Da, about 2,000,000 Da to about5,000,000 Da, about 2,500,000 Da to about 5,000,000 Da, about 2,000,000Da to about 3,000,000 Da, about 2,500,000 Da to about 3,500,000 Da, orabout 2,000,000 Da to about 4,000,000 Da. In some embodiments, acomposition comprises an uncrosslinked hyaluronan where theuncrosslinked hyaluronan has a mean molecular weight of, e.g., greaterthan 2,000,000 Da and less than about 3,000,000 Da, greater than2,000,000 Da and less than about 3,500,000 Da, greater than 2,000,000 Daand less than about 4,000,000 Da, greater than 2,000,000 Da and lessthan about 4,500,000 Da, greater than 2,000,000 Da and less than about5,000,000 Da.

A composition disclosed herein comprises a gel phase including a silkfibroin hydrogel component or particle and matrix polymer hydrogelcomponent or particle. In some embodiments, the percent amount of silkfibroin hydrogel present in a composition relative to matrix polymerhydrogel is from about 0.1% (v/v) to about 25% (v/v). In someembodiments, the percent amount of matrix polymer hydrogel present in acomposition relative to silk fibroin hydrogel is from about 99.9% (v/v)to about 75% (v/v). In some embodiments, the ratio of silk fibroinhydrogel to matrix polymer hydrogel in the gel phase of a compositioncomprises, e.g., about 0.1% (v/v) silk fibroin hydrogel and about 99.9%(v/v) matrix polymer hydrogel, about 1% (v/v) silk fibroin hydrogel andabout 99% (v/v) matrix polymer hydrogel, about 5% (v/v) silk fibroinhydrogel and about 95% (v/v) matrix polymer hydrogel, about 10% (v/v)silk fibroin hydrogel and about 90% (v/v) matrix polymer hydrogel, about15% (v/v) silk fibroin hydrogel and about 85% (v/v) matrix polymerhydrogel, about 20% (v/v) silk fibroin hydrogel and about 80% (v/v)matrix polymer hydrogel, or about 25% (v/v) silk fibroin hydrogel andabout 75% (v/v) matrix polymer hydrogel.

A composition disclosed herein may comprise a gel phase where the silkfibroin hydrogel component and matrix polymer hydrogel component areprocessed separately. The resulting processed hydrogel materials, e.g.,hydrogel particles of both types, are then mixed together, such as,e.g., after a milling step and/or after re-homogenization in a carrierphase, to form the final composition. In addition, a matrix polymer maybe initially mixed with depolymerized silk fibroin solution, withsubsequent polymerization occurring only after the completion of themixing step to form an integrated matrix polymer/silk fibroin compositehydrogel. Similarly, the silk fibroin and matrix polymers may be linkedtogether to form a hydrogel composite that is then subsequentlyprocessed into the gel phase of the composition. Such linkage can occurby a typical cross linking method or by linking the matrix polymer tothe silk fibroin hydrogel via a peptide linker disclosed herein, suchas, e.g., a five-amino acid peptide “tail” and synthetic molecule. Asdisclosed herein, a composition may comprise a gel phase that comprisesboth separately processed hydrogel components as well as particles ofhydrogel composites.

As a non-limiting example, a solution comprising about 1% to about 30%depolymerized silk fibroin may be mixed with about 6 mg/g to about 30mg/g of hyaluronan having a degree of cross linking of from 0 to about17% where the percent weight of the silk fibroin component is from about1% to about 75%. As another non-limiting example, hydrogel particlescomprising from about 1% to about 8% silk fibroin are mixed withhydrogel particles comprising about 6 mg/g to about 30 mg/g ofhyaluronan having a degree of cross linking of from 0 to about 17% wherethe percent weight of the silk fibroin component is from about 1% toabout 75%. As yet another non-limiting example, a hydrogel compositioncomprising hydrogel particles comprising from about 1% to about 8% silkfibroin mixed together with a carrier phase (about 20% (v/v) to about50% (v/v)) is mixed with a hydrogel composition comprising hydrogelparticles comprising about 6 mg/g to about 30 mg/g of hyaluronan havinga degree of cross linking of from 0 to about 17% where the percentweight of the silk fibroin component is from about 1% to about 75%.

Aspects of the present specification provide, in part, a compositioncomprising a silk fibroin hydrogel component or particle and matrixpolymer hydrogel component or particle having opacity. Opacity is themeasure of impenetrability to electromagnetic or other kinds ofradiation, especially visible light. An opaque object is neithertransparent (allowing all light to pass through) nor translucent(allowing some light to pass through). In certain applications, it wouldbe an advantage to have an opaque composition. For example, inapplications where a composition disclosed herein is administered to asuperficial region, an opaque composition provides coloration andappearance of the overlying skin.

In an embodiment, a composition comprising a silk fibroin hydrogel and apolymer matrix is optically opaque. In some embodiments, a compositioncomprising a silk fibroin hydrogel and a polymer matrix transmits, e.g.,about 5% of the light to about 70% of the light. In some preferred someembodiments, a composition comprising a silk fibroin hydrogel and apolymer matrix transmits, e.g., about 10% of the light to about 65% ofthe light. In the most preferred some embodiments, a compositioncomprising a silk fibroin hydrogel and a polymer matrix transmits, e.g.,about 15% of the light to about 60% of the light.

In some embodiments, a composition comprising a silk fibroin hydrogeland a polymer matrix exhibits, e.g., about 5% to about 100% reduction intyndalling. In some preferred some embodiments, a composition comprisinga silk fibroin hydrogel and a polymer matrix exhibits, e.g., about 10%to about 95% reduction in tyndalling. In the most preferred someembodiments, a composition comprising a silk fibroin hydrogel and apolymer matrix exhibits, e.g., about 15% to about 90% reduction intyndalling.

Aspects of the present specification provide, in part, a compositiondisclosed herein exhibiting a dynamic viscosity. Viscosity is resistanceof a fluid to shear or flow caused by either shear stress or tensilestress. Viscosity describes a fluid's internal resistance to flow causedby intermolecular friction exerted when layers of fluids attempt toslide by one another and may be thought of as a measure of fluidfriction. The less viscous the fluid, the greater its ease of movement(fluidity).

Viscosity can be defined in two ways; dynamic viscosity (μ, although ηis sometimes used) or kinematic viscosity (ν). Dynamic viscosity, alsoknown as absolute or complex viscosity, is the tangential force per unitarea required to move one horizontal plane with respect to the other atunit velocity when maintained a unit distance apart by the fluid. The SIphysical unit of dynamic viscosity is the Pascal-second (Pa·s), which isidentical to N·m-2·s. Dynamic viscosity can be expressed as τ=μ dvx/dz,where τ=shearing stress, μ=dynamic viscosity, and dvx/dz is the velocitygradient over time. For example, if a fluid with a viscosity of one Pa·sis placed between two plates, and one plate is pushed sideways with ashear stress of one Pascal, it moves a distance equal to the thicknessof the layer between the plates in one second. Dynamic viscositysymbolize by is also used, is measured with various types of rheometers,devices used to measure the way in which a liquid, suspension or slurryflows in response to applied forces.

Kinematic viscosity (ν) is the ratio of dynamic viscosity to density, aquantity in which no force is involved and is defined as follows: ν=μ/ρ,where μ is the dynamic viscosity ρ is density with the SI unit of kg/m³.Kinematic viscosity is usually measured by a glass capillary viscometeras has an SI unit of m²/s.

The viscosity of a fluid is highly temperature dependent and for eitherdynamic or kinematic viscosity to be meaningful, the referencetemperature must be quoted. For the viscosity values disclosed herein, adynamic viscosity is measured at 1 Pa with a cone/plane geometry 2° /40cm and a temperature of 20° C. Examples of the dynamic viscosity ofvarious fluids at 20° C. is as follows: water is about 1.0×10⁻³ Pa·s,blood is about 3-4×10⁻³ Pa·s, vegetable oil is about 60-85×10⁻³ Pa·s,motor oil SE 30 is about 0.2 Pa·s, glycerin is about 1.4 Pa·s, maplesyrup is about 2-3 Pa·s, honey is about 10 Pa·s, chocolate syrup isabout 10-25 Pa·s, peanut butter is about 150-250 Pa·s, lard is about1,000 Pa·s, vegetable shortening is about 1,200 Pa·s, and tar is about30,000 Pa·s.

In some embodiments, a composition disclosed herein exhibits a dynamicviscosity of, e.g., about 10 Pa·s to about 1,200 Pa·s. In some preferredsome embodiments, a composition disclosed herein exhibits a dynamicviscosity of, e.g., about 20 Pa·s to about 1,100 Pa·s. In the mostpreferred some embodiments, a composition disclosed herein exhibits adynamic viscosity of, e.g., about 30 Pa·s to about 1,000 Pa·s.

Aspects of the present specification provide, in part, a compositiondisclosed herein is injectable. As used herein, the term “injectable”refers to a material having the properties necessary to administer thecomposition into a skin region of an individual using an injectiondevice with a fine needle. As used herein, the term “fine needle” refersto a needle that is 27 gauge or smaller. Injectability of a compositiondisclosed herein can be accomplished by sizing the hydrogel particles asdiscussed above.

In some embodiments, a composition disclosed herein is injectablethrough a fine needle. In other some embodiments, a compositiondisclosed herein is injectable through a needle of, e.g., about 27gauge, about 30 gauge, or about 32 gauge. In yet other some embodiments,a composition disclosed herein is injectable through a needle of, e.g.,27 gauge or smaller, 30 gauge or smaller, or 32 gauge or smaller. Instill other some embodiments, a composition disclosed herein isinjectable through a needle of, e.g., about 27 gauge to about 32 gauge.

In some embodiments, a composition disclosed herein can be injected withan extrusion force of about 60 N to about 5 N or less. In some preferredsome embodiments, a composition disclosed herein can be injected with anextrusion force of about 55 N to about 10 N or less. In the mostpreferred some embodiments, a composition disclosed herein can beinjected with an extrusion force of about 50 N to about 15 N or less.

Aspects of the present specification provide, in part, a compositiondisclosed herein exhibits cohesiveness. Cohesion or cohesive attraction,cohesive force, or compression force is a physical property of amaterial, caused by the intermolecular attraction between like-moleculeswithin the material that acts to unite the molecules. A compositionshould be sufficiently cohesive as to remain localized to a site ofadministration. Additionally, in certain applications, a sufficientcohesiveness is important for a composition to retain its shape, andthus functionality, in the event of mechanical load cycling. As aresult, in one embodiment, a composition exhibits strong cohesiveattraction, on par with water. In another embodiment, a compositionexhibits low cohesive attraction. In yet another embodiment, acomposition exhibits sufficient cohesive attraction to remain localizedto a site of administration. In still another embodiment, a compositionexhibits sufficient cohesive attraction to retain its shape. In afurther embodiment, a composition exhibits sufficient cohesiveattraction to retain its shape and functionality.

In some embodiments, a composition disclosed herein has a compressionforce of about 10 grams-force to about 3000 grams-force. In somepreferred some embodiments, a composition disclosed herein has acompression force of about 20 grams-force to about 2000 grams-force. Inthe most preferred of this embodiment, a composition disclosed hereinhas a compression force of about 30 grams-force to about 1000grams-force.

Aspects of the present specification provide, in part, a method oftreating a soft tissue condition of an individual by administering acomposition disclosed herein. As used herein, the term “treating,”refers to reducing or eliminating in an individual a cosmetic orclinical symptom of a soft tissue condition characterized by a softtissue imperfection, defect, disease, and/or disorder; or delaying orpreventing in an individual the onset of a cosmetic or clinical symptomof a condition characterized by a soft tissue imperfection, defect,disease, and/or disorder. For example, the term “treating” can meanreducing a symptom of a condition characterized by a soft tissue defect,disease, and/or disorder by, e.g., at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90% or at least 100%. The effectiveness of a compound disclosed hereinin treating a condition characterized by a soft tissue defect, disease,and/or disorder can be determined by observing one or more cosmetic,clinical symptoms, and/or physiological indicators associated with thecondition. An improvement in a soft tissue defect, disease, and/ordisorder also can be indicated by a reduced need for a concurrenttherapy. Those of skill in the art will know the appropriate symptoms orindicators associated with specific soft tissue defect, disease, and/ordisorder and will know how to determine if an individual is a candidatefor treatment with a compound or composition disclosed herein.

A composition or compound is administered to an individual. Anindividual is typically a human being. Typically, any individual who isa candidate for a conventional procedure to treat a soft tissuecondition is a candidate for a method disclosed herein. In addition, thepresently disclosed compositions and methods may apply to individualsseeking a small/moderate enlargement, shape change or contour alterationof a body part or region, which may not be technically possible oraesthetically acceptable with existing soft tissue implant technology.Pre-operative evaluation typically includes routine history and physicalexamination in addition to thorough informed consent disclosing allrelevant risks and benefits of the procedure.

The composition and methods disclosed herein are useful in treating asoft tissue condition. A soft tissue condition includes, withoutlimitation, a soft tissue imperfection, defect, disease, and/ordisorder. Non-limiting examples of a soft tissue condition includebreast imperfection, defect, disease and/or disorder, such as, e.g., abreast augmentation, a breast reconstruction, mastopexy, micromastia,thoracic hypoplasia, Poland's syndrome, defects due to implantcomplications like capsular contraction and/or rupture; a facialimperfection, defect, disease or disorder, such as, e.g., a facialaugmentation, a facial reconstruction, Parry-Romberg syndrome, lupuserythematosus profundus, dermal divots, sunken checks, thin lips, nasalimperfections or defects, retro-orbital imperfections or defects, afacial fold, line and/or wrinkle like a glabellar line, a nasolabialline, a perioral line, and/or a marionette line, and/or other contourdeformities or imperfections of the face; a neck imperfection, defect,disease or disorder; a skin imperfection, defect, disease and/ordisorder; other soft tissue imperfections, defects, diseases and/ordisorders, such as, e.g., an augmentation or a reconstruction of theupper arm, lower arm, hand, shoulder, back, torso including abdomen,buttocks, upper leg, lower leg including calves, foot including plantarfat pad, eye, genitals, or other body part, region or area, or a diseaseor disorder affecting these body parts, regions or areas; urinaryincontinence, fecal incontinence, other forms of incontinence; andgastroesophageal reflux disease (GERD).

The amount of a composition used with any of the methods as disclosedherein will typically be determined based on the alteration and/orimprovement desired, the reduction and/or elimination of a soft tissuecondition symptom desired, the clinical and/or cosmetic effect desiredby the individual and/or physician, and the body part or region beingtreated. The effectiveness of composition administration may bemanifested by one or more of the following clinical and/or cosmeticmeasures: altered and/or improved soft tissue shape, altered and/orimproved soft tissue size, altered and/or improved soft tissue contour,altered and/or improved tissue function, tissue ingrowth support and/ornew collagen deposition, sustained engraftment of composition, improvedpatient satisfaction and/or quality of life, and decreased use ofimplantable foreign material.

For example, for breast augmentation procedures, effectiveness of thecompositions and methods may be manifested by one or more of thefollowing clinical and/or cosmetic measures: increased breast size,altered breast shape, altered breast contour, sustained engraftment,reduction in the risk of capsular contraction, decreased rate ofliponecrotic cyst formation, improved patient satisfaction and/orquality of life, and decreased use of breast implant.

As another example, effectiveness of the compositions and methods intreating a facial soft tissue may be manifested by one or more of thefollowing clinical and/or cosmetic measures: increased size, shape,and/or contour of facial feature like increased size, shape, and/orcontour of lip, cheek or eye region; altered size, shape, and/or contourof facial feature like altered size, shape, and/or contour of lip, cheekor eye region shape; reduction or elimination of a wrinkle, fold or linein the skin; resistance to a wrinkle, fold or line in the skin;rehydration of the skin; increased elasticity to the skin; reduction orelimination of skin roughness; increased and/or improved skin tautness;reduction or elimination of stretch lines or marks; increased and/orimproved skin tone, shine, brightness and/or radiance; increased and/orimproved skin color, reduction or elimination of skin paleness;sustained engraftment of composition; decreased side effects; improvedpatient satisfaction and/or quality of life.

The amount of a composition used with any of the methods disclosedherein will typically be a therapeutically effective amount. As usedherein, the term “therapeutically effective amount” is synonymous with“effective amount”, “therapeutically effective dose”, and/or “ effectivedose” and refers to the amount of compound that will elicit thebiological, cosmetic or clinical response being sought by thepractitioner in an individual in need thereof. As a non-limitingexample, an effective amount is an amount sufficient to achieve one ormore of the clinical and/or cosmetic measures disclosed herein. Theappropriate effective amount to be administered for a particularapplication of the disclosed methods can be determined by those skilledin the art, using the guidance provided herein. For example, aneffective amount can be extrapolated from in vitro and in vivo assays asdescribed in the present specification. One skilled in the art willrecognize that the condition of the individual can be monitoredthroughout the course of therapy and that the effective amount of acomposition disclosed herein that is administered can be adjustedaccordingly.

In some embodiments, the amount of a composition administered is, e.g.,0.01 g, 0.05 g, 0.1 g, 0.5 g, 1 g, 5 g, 10 g, 20 g, 30 g, 40 g, 50 g, 60g, 70 g, 80 g, 90 g, 100 g, 150 g, or 200 g. In other some embodiments,the amount of a composition administered is, e.g., about 0.01 g to about0.1 g, about 0.1 g to about 1 g, about 1 g to about 10 g, about 10 g toabout 100 g, or about 50 g to about 200 g. In yet other someembodiments, the amount of a composition administered is, e.g., 0.01 mL,0.05 mL, 0.1 mL, 0.5 mL, 1 mL, 5 mL, 10 mL, 20 mL, 30 mL, 40 mL, 50 mL,60 mL, 70 g, 80 mL, 90 mL, 100 mL, 150 mL, or 200 mL. In other someembodiments, the amount of a composition administered is, e.g., about0.01 mL to about 0.1 mL, about 0.1 mL to about 1 mL, about 1 mL to about10 mL, about 10 mL to about 100 mL, or about 50 mL to about 200 mL.

Aspects of some embodiments provide, in part, administering acomposition disclosed herein. As used herein, the term “administering”means any delivery mechanism that provides a composition disclosedherein to an individual that potentially results in a clinically,therapeutically, or experimentally beneficial result. The actualdelivery mechanism used to administer a composition to an individual canbe determined by a person of ordinary skill in the art by taking intoaccount factors, including, without limitation, the type of skincondition, the location of the skin condition, the cause of the skincondition, the severity of the skin condition, the degree of reliefdesired, the duration of relief desired, the particular compositionused, the rate of excretion of the particular composition used, thepharmacodynamics of the particular composition used, the nature of theother compounds included in the particular composition used, theparticular route of administration, the particular characteristics,history and risk factors of the individual, such as, e.g., age, weight,general health and the like, or any combination thereof. In someembodiments, a composition disclosed herein is administered to a skinregion of an individual by injection.

The route of administration of composition administered to an individualpatient will typically be determined based on the cosmetic and/orclinical effect desired by the individual and/or physician and the bodypart or region being treated. A composition disclosed herein may beadministered by any means known to persons of ordinary skill in the artincluding, without limitation, syringe with needle, catheter, topically,or by direct surgical implantation. The composition disclosed herein canbe administered into a skin region such as, e.g., a dermal region or ahypodermal region. In addition, a composition disclosed herein can beadministered once, or over a plurality of times. Ultimately, the timingused will follow quality care standards.

For a breast soft tissue replacement procedure, the route ofadministration may include axillary, periareolar, and/or inframammaryroutes. Alternatively or in addition, a composition may be deliveredthrough a transaxillary endoscopic subpectoral approach. For a facialsoft tissue replacement procedure, the route of administration can befrontal, temporal, zygomatic, periocular, amdibula, perioral or chinroutes. In urinary incontinence procedures, the route of administrationmay include transurethral or periurethral routes. Alternatively or inaddition, administration may be delivered via an antegrade route. Theroutes discussed herein do not exclude the use of multiple routes toachieve the desired clinical effect.

Aspects of some embodiments provide, in part, a dermal region. As usedherein, the term “dermal region” refers to the region of skin comprisingthe epidermal-dermal junction and the dermis including the superficialdermis (papillary region) and the deep dermis (reticular region). Theskin is composed of three primary layers: the epidermis, which provideswaterproofing and serves as a barrier to infection; the dermis, whichserves as a location for the appendages of skin; and the hypodermis(subcutaneous adipose layer). The epidermis contains no blood vessels,and is nourished by diffusion from the dermis. The main type of cellswhich make up the epidermis are keratinocytes, melanocytes, Langerhanscells and Merkels cells.

The dermis is the layer of skin beneath the epidermis that consists ofconnective tissue and cushions the body from stress and strain. Thedermis is tightly connected to the epidermis by a basement membrane. Italso harbors many Mechanoreceptor/nerve endings that provide the senseof touch and heat. It contains the hair follicles, sweat glands,sebaceous glands, apocrine glands, lymphatic vessels and blood vessels.The blood vessels in the dermis provide nourishment and waste removalfrom its own cells as well as from the Stratum basale of the epidermis.The dermis is structurally divided into two areas: a superficial areaadjacent to the epidermis, called the papillary region, and a deepthicker area known as the reticular region.

The papillary region is composed of loose areolar connective tissue. Itis named for its fingerlike projections called papillae that extendtoward the epidermis. The papillae provide the dermis with a “bumpy”surface that interdigitates with the epidermis, strengthening theconnection between the two layers of skin. The reticular region liesdeep in the papillary region and is usually much thicker. It is composedof dense irregular connective tissue, and receives its name from thedense concentration of collagenous, elastic, and reticular fibers thatweave throughout it. These protein fibers give the dermis its propertiesof strength, extensibility, and elasticity. Also located within thereticular region are the roots of the hair, sebaceous glands, sweatglands, receptors, nails, and blood vessels. Tattoo ink is held in thedermis. Stretch marks from pregnancy are also located in the dermis.

The hypodermis lies below the dermis. Its purpose is to attach thedermal region of the skin to underlying bone and muscle as well assupplying it with blood vessels and nerves. It consists of looseconnective tissue and elastin. The main cell types are fibroblasts,macrophages and adipocytes (the hypodermis contains 50% of body fat).Fat serves as padding and insulation for the body.

In some embodiments, a composition disclosed herein is administered to askin region of an individual by injection into a dermal region or ahypodermal region. In some embodiments, a composition disclosed hereinis administered to a dermal region of an individual by injection into,e.g., an epidermal-dermal junction region, a papillary region, areticular region, or any combination thereof.

Aspects of the present specification disclose, in part, a method oftreating a soft tissue condition of an individual, the method comprisingthe steps of administering a composition disclosed herein to a site ofthe soft tissue condition of the individual, wherein the administrationof the composition improves the soft tissue condition, thereby treatingthe soft tissue condition. In some embodiments, a soft tissue conditionis a breast tissue condition, a facial tissue condition, a neckcondition, a skin condition, an upper arm condition, a lower armcondition, a hand condition, a shoulder condition, a back condition, atorso including abdominal condition, a buttock condition, an upper legcondition, a lower leg condition including calf condition, a footcondition including plantar fat pad condition, an eye condition, agenital condition, or a condition effecting another body part, region orarea.

Some embodiments relate at least in part to a method of treating a skincondition comprises the step of administering to an individual sufferingfrom a skin condition a composition disclosed herein, wherein theadministration of the composition improves the skin condition, therebytreating the skin condition. In some embodiments, a skin condition is amethod of treating skin dehydration comprises the step of administeringto an individual suffering from skin dehydration a composition disclosedherein, wherein the administration of the composition rehydrates theskin, thereby treating skin dehydration. In some embodiments, a methodof treating a lack of skin elasticity comprises the step ofadministering to an individual suffering from a lack of skin elasticitya composition disclosed herein, wherein the administration of thecomposition increases the elasticity of the skin, thereby treating alack of skin elasticity. In yet some embodiments, a method of treatingskin roughness comprises the step of administering to an individualsuffering from skin roughness a composition disclosed herein, whereinthe administration of the composition decreases skin roughness, therebytreating skin roughness. In still some embodiments, a method of treatinga lack of skin tautness comprises the step of administering to anindividual suffering from a lack of skin tautness a compositiondisclosed herein, wherein the administration of the composition makesthe skin tauter, thereby treating a lack of skin tautness.

In some embodiments, a method of treating a skin stretch line or markcomprises the step of administering to an individual suffering from askin stretch line or mark a composition disclosed herein, wherein theadministration of the composition reduces or eliminates the skin stretchline or mark, thereby treating a skin stretch line or mark. In someembodiments, a method of treating skin paleness comprises the step ofadministering to an individual suffering from skin paleness acomposition disclosed herein, wherein the administration of thecomposition increases skin tone or radiance, thereby treating skinpaleness. In some embodiments, a method of treating skin wrinklescomprises the step of administering to an individual suffering from skinwrinkles a composition disclosed herein, wherein the administration ofthe composition reduces or eliminates skin wrinkles, thereby treatingskin wrinkles. In yet some embodiments, a method of treating skinwrinkles comprises the step of administering to an individual acomposition disclosed herein, wherein the administration of thecomposition makes the skin resistant to skin wrinkles, thereby treatingskin wrinkles.

Aspects of the present specification provide, in part, administration ofa composition disclosed herein wherein such administration promotes newcollagen deposition. The compositions comprising a silk fibroin hydrogelcomponent or particle and matrix polymer hydrogel component or particlesupport tissue ingrowth and new deposition of collagen (Example 21).

In an embodiment, administration of a composition comprising a silkfibroin hydrogel component and a matrix polymer hydrogel component asdisclosed herein increases new collagen deposition. In some embodiments,administration of a composition comprising a silk fibroin hydrogelcomponent and a matrix polymer hydrogel component as disclosed hereinincreases new collagen deposition by about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, orabout 100%, relative to a the same or similar composition comprising thematrix polymer hydrogel component, but lacking the silk fibroin hydrogelcomponent. In other some embodiments, administration of a compositioncomprising a silk fibroin hydrogel component and a matrix polymerhydrogel component as disclosed herein increases new collagen depositionby at least 25%, at least 50%, at least 75%, at least 100%, at least125%, at least 150%, at least 175%, at least 200%, at least 225%, atleast 250%, at least 275%, or at least 300%, relative to a the same orsimilar composition comprising the matrix polymer hydrogel component,but lacking the silk fibroin hydrogel component. In yet other someembodiments, administration of a composition comprising a silk fibroinhydrogel component and a matrix polymer hydrogel component as disclosedherein increases new collagen deposition by about 10% to about 100%,about 50% to about 150%, about 100% to about 200%, about 150% to about250%, about 200% to about 300%, about 350% to about 450%, about 400% toabout 500%, about 550% to about 650%, about 600% to about 700%, relativeto a the same or similar composition comprising the matrix polymerhydrogel component, but lacking the silk fibroin hydrogel component.

As used herein, the terms “adipose tissue,” “fat,” “fat tissue”, or“fatty tissue” include loose fibrous connective tissue comprising fatcells (adipocytes) and multiple types of regenerative cells, and maycomprise brown and/or white adipose tissue taken from any body site,such as, e.g., subcutaneous, omental/visceral, interscapular, ormediastinal. It may be obtained from any organism having adipose tissue,or the adipose tissue used may be from a primary cell culture or animmortalized cell line.

Adipose tissue may be collected from the same individual who isundergoing the soft tissue replacement procedure (autograft), from adonor individual who is not the same individual as the one undergoingthe soft tissue replacement procedure (allograft), or from an animalsource (xenograft). As used herein, the term “autotransplantation”refers to the transplantation of organs, tissues, or cells from one partof the body to another part in the same individual, i.e., the donor andrecipient are the same individual. Tissue transplanted by such“autologous” procedures is referred to as an autograft orautotransplant. As used herein, the term “allotransplantation” refers tothe transplantation of organs, tissues, or cells from a donor to arecipient, where the donor and recipient are different individuals, butof the same species. Tissue transplanted by such “allologous” proceduresis referred to as an allograft or allotransplant. As used herein, theterm “xenotransplantation” refers to the transplantation of organs,tissues, or cells from a donor to a recipient, where the donor is of adifferent species as the recipient. Tissue transplanted by such“xenologous” procedures is referred to as a xenograft or xenotransplant.

Adipose tissue can be collected by any procedure that can harvestadipose tissue useful for the compositions and methods disclosed herein,including, without limitation a liposuction (lipoplasty) procedure or alipectomy procedure. Procedures useful for collecting adipose tissueshould minimize the trauma and manipulation associated with adiposetissue removed. Adipose tissue may be harvested from any suitableregion, including, without limitation, a mammary region, an abdominalregion, a thigh region, a flank region, a gluteal region, a trochanterregion, or a gonadal region. Procedures useful for collecting adiposetissue are well known to a person of ordinary skill in the art. Theselected procedures may be performed concomitantly with liposculpture.

A liposuction procedure harvests adipose tissue by aspirating the tissueusing a cannula. The cannula may be connected to a syringe for manualaspiration or to a power assisted suction device, like an aspirator,adapted to collect the adipose tissue into a vacuum bottle. Aliposuction procedure does not maintain an intact blood supply of theharvested tissue. The syringe may be a 10, 20 or 60 mL syringe fittedwith a 12 or 14 gauge cannula. Non-limiting examples of liposuctionprocedures include suction-assisted liposuction (SAL),ultrasound-assisted liposuction (UAL), power-assisted liposuction (PAL),twin-cannula (assisted) liposuction (TCAL or TCL), or externalultrasound-assisted liposuction (XUAL or EUAL), or water-assistedliposuction (WAL). In addition, the liposuction procedures listed abovecan be used with any of the following procedures that vary the amount offluid injected during the procedure, such as, e.g., dry liposuction, wetliposuction, super-wet liposuction, tumescent liposuction, orlaser-assisted liposuction. An autologous soft tissue transfer proceduretypically uses adipose tissue collected from a liposuction procedure.

Although the harvested tissue may be used directly to make the disclosedcompositions, it is more typically processed to purify and/or enrich forhealthy adipocytes and regenerative cells. For example, the harvestedadipose tissue may be separated from any debris and/or contaminants suchas, e.g., blood, serum, proteases, lipases, lipids and other oils,and/or other bodily fluids; tumescent fluid and/or other materials usedin the liposuction procedure; and/or other impurities suctioned duringthe procedure. Methods useful in separating debris and/or contaminantsfrom adipose tissue useful to make the disclosed compositions,including, without limitation, centrifugation, sedimentation,filtration, and/or absorption. In addition, or alternatively, theharvested adipose tissue may be processed by washing is a physiologicalbuffer like saline to remove any debris and/or contaminants.

A lipectomy procedure harvests adipose tissue by surgical excision froma donor site in a manner that minimizes damage to the blood supply ofthe tissue using standard surgical operative procedures. This harvestedtissue is then implanted into the region needing the soft tissuereplacement. A tissue flap or tissue graft procedure typically usesadipose tissue collected from a lipectomy procedure. A tissue flap is asection of living tissue that maintained its blood supply as the tissueis moved from one area of the body to another.

A local flap uses a piece of skin and underlying tissue that lieadjacent to the wound, including adipose tissue. The flap remainsattached at one end so that it continues to be nourished by its originalblood supply, and is repositioned over the wounded area. A regional flapuses a section of tissue that is attached by a specific blood vessel.When the flap is lifted, it needs only a very narrow attachment to theoriginal site to receive its nourishing blood supply from the tetheredartery and vein. A musculocutaneous flap, also called a muscle and skinflap, is used when the area to be covered needs more bulk and a morerobust blood supply. Musculocutaneous flaps are often used in breastreconstruction to rebuild a breast after mastectomy. As an example, thetransverse rectus abdominus myocutaneous) flap (TRAM flap) is a tissueflap procedure that uses muscle, fat and skin from an abdomen to createa new breast mound after a mastectomy. This type of flap remains“tethered” to its original blood supply. In a bone/soft tissue flap,bone, along with the overlying skin, is transferred to the wounded area,carrying its own blood supply.

Typically, a wound that is wide and difficult or impossible to closedirectly may be treated with a skin graft. A skin graft is a patch ofhealthy skin that is taken from one area of the body, called the “donorsite,” and used to cover another area where skin is missing or damaged.There are three basic types of skin grafts. A split-thickness skingraft, commonly used to treat burn wounds, uses only the layers of skinclosest to the surface. A full-thickness skin graft might be used totreat a burn wound that is deep and large, or to cover jointed areaswhere maximum skin elasticity and movement are desired. As its nameimplies, a full-thickness (all layers) section of skin from the donorsite are lifted. A composite graft is used when the wound to be coveredneeds more underlying support, as with skin cancer on the nose. Acomposite graft requires lifting all the layers of skin, adipose tissue,and sometimes the underlying cartilage from the donor site.

The amount of adipose tissue collected will typically vary fromindividual to individual and can depend on a number of factorsincluding, but not limited to, amount of adipose tissue required for thesoft tissue replacement method, aesthetic expectations, age, bodyhabitus, coagulation profile, hemodynamic stability, co-morbidities, andphysician preference. A liposuction procedure may harvest from about 1mL to about 1500 mL of adipose tissue. A lipectomy procedure typicallyharvests about 1 g to about 5,000 g.

Adipose tissue comprises multiple types of regenerative cells. As usedherein, the term “regenerative cell” refers to any cells that cause orcontribute to complete or partial regeneration, restoration, orsubstitution of structure or function of an organ, tissue, orphysiologic unit or system to thereby provide a therapeutic, structuralor cosmetic benefit. Examples of regenerative cells include stem cells,progenitor cells, and precursor cells.

As used herein, the term “stem cell” refers to a multipotentregenerative cell with the potential to differentiate into a variety ofother cell types that perform one or more specific functions and has theability to self-renew. Some of the stem cells disclosed herein may bepluripotent. Exemplary examples of stem cells include, withoutlimitation, adipose-derived stem cells (ASCs; adipose-derived stromalcells), endothelial-derived stem cells (ESCs), hemopoietic stem cells(HSCs), and mesenchyma stem cells (MSCs). Examples of differentiationinclude angiogenesis, neovascularization, adipogenesis and collagenesis

As used herein, the term “progenitor cell” refers to an oligopotentregenerative cell with the potential to differentiate into more than onecell type, or a unipotent regenerative cell with the potential todifferentiate into only a single cell type, that perform(s) one or morespecific functions and has limited or no ability to self-renew.Exemplary examples of progenitor cells include, without limitation,endothelial progenitor cells, keratinocytes, monoblasts, myoblasts, andpericytes.

As used herein, the term “precursor cell” refers to a unipotentregenerative cell with the potential to differentiate into one cell typethat performs one or more specific functions and may retain extensiveproliferative capacity that enables the cells to proliferate underappropriate conditions. Exemplary examples of precursor cells include,without limitation, adipoblast (lipoblast or preadipocytes),de-differentiated adipocytes, angioblasts, endothelial precursor cells,fibroblasts, lymphoblasts, and macrophages.

A hydrogel composition disclosed herein may enhance differentiation ofthe multiple regenerative cells from the adipose tissue. In oneembodiment, a hydrogel composition disclosed herein enhancesdifferentiation of the multiple regenerative cells from the adiposetissue as compared to adipose tissue alone. In some embodiments, ahydrogel composition disclosed herein enhances differentiation of themultiple regenerative cells from the adipose tissue by at least about50% at least about 100%, at least about 150%, at least about 200%, atleast 250%, at least about 300%, at least about 350%, at least about400%, at least about 450%, at least about 500%, at least about 750%, orat least about 1000% as compared to adipose tissue alone. In someembodiments, a hydrogel composition disclosed herein enhancesdifferentiation of the multiple regenerative cells from the adiposetissue by about 50% to about 250%, about 50% to about 500%, about 50% toabout 1000%, about 100% to about 300%, about 100% to about 500%, about100% to about 1000%, about 150% to about 400%, about 150% to about 600%,about 150% to about 1000%, about 200% to about 500%, about 200% to about700%, or about 200% to about 1000% as compared to adipose tissue alone.

In another embodiment, a hydrogel composition disclosed herein enhancesdifferentiation of the multiple regenerative cells from the adiposetissue as compared to adipose tissue with a hydrogel composition that issubstantially identical except that the hyaluronic acid component andthe silk fibroin component are not crosslinked. In some embodiments, ahydrogel composition disclosed herein enhances differentiation of themultiple regenerative cells from the adipose tissue by at least about50% at least about 100%, at least about 150%, at least about 200%, atleast 250%, at least about 300%, at least about 350%, at least about400%, at least about 450%, at least about 500%, at least about 750%, orat least about 1000% as compared to adipose tissue with a hydrogelcomposition that is substantially identical except that the hyaluronicacid component and the silk fibroin component are not crosslinked. Insome embodiments, a hydrogel composition disclosed herein enhancesdifferentiation of the multiple regenerative cells from the adiposetissue by about 50% to about 250%, about 50% to about 500%, about 50% toabout 1000%, about 100% to about 300%, about 100% to about 500%, about100% to about 1000%, about 150% to about 400%, about 150% to about 600%,about 150% to about 1000%, about 200% to about 500%, about 200% to about700%, or about 200% to about 1000% as compared to adipose tissue with ahydrogel composition that is substantially identical except that thehyaluronic acid component and the silk fibroin component are notcrosslinked. In some embodiments, a hydrogel composition that issubstantially identical to a hydrogel composition disclosed hereinexcept that the hyaluronic acid component and the silk fibroin componentare not crosslinked comprises hyaluronic acid at a concentration ofabout 16 mg/mL or about 24 mg/mL and water.

In yet another embodiment, a hydrogel composition disclosed hereinenhances differentiation of the multiple regenerative cells from theadipose tissue as compared to adipose tissue with a hydrogel compositionthat is substantially identical except that the silk fibroin componentis absent. In some embodiments, a hydrogel composition disclosed hereinenhances differentiation of the multiple regenerative cells from theadipose tissue by at least about 50% at least about 100%, at least about150%, at least about 200%, at least 250%, at least about 300%, at leastabout 350%, at least about 400%, at least about 450%, at least about500%, at least about 750%, or at least about 1000% as compared toadipose tissue with a hydrogel composition that is substantiallyidentical except that the silk fibroin component is absent. In someembodiments, a hydrogel composition disclosed herein enhancesdifferentiation of the multiple regenerative cells from the adiposetissue by about 50% to about 250%, about 50% to about 500%, about 50% toabout 1000%, about 100% to about 300%, about 100% to about 500%, about100% to about 1000%, about 150% to about 400%, about 150% to about 600%,about 150% to about 1000%, about 200% to about 500%, about 200% to about700%, or about 200% to about 1000% as compared to adipose tissue with ahydrogel composition that is substantially identical except that thesilk fibroin component is absent. In some embodiments, a hydrogelcomposition that is substantially identical to a hydrogel compositiondisclosed herein except that the silk fibroin component is absentcomprises hyaluronic acid at a concentration of about 16 mg/mL or about24 mg/mL and water.

Harvested adipose tissue useful in compositions of some embodiments canbe supplemented with regenerative cells such as, e.g., stem cells,progenitor cells, and precursor cells. Regenerative cells may promotenew blood vessel formation, diminish necrosis, and/or promote asupportive microenvironment in the transplanted tissue, therebyimproving survivability of the transplanted tissue. Regenerative cellscan be obtained from a variety of sources. For example, adipose tissueis rich in regenerative cells that have the ability to restore andreconstruct various soft tissue defects in response to localdifferentiation clues from the recipient site. As such, a portion of thecollected adipose tissue may be further processed in order to purifyregenerative cells that can then be added back to the remainder of theharvested adipose tissue in order to enrich this material for thesecells. Exemplary methods describing such cell enrichment procedures canbe found in, e.g., Hedrick and Fraser, Methods of Using AdiposeTissue-Derived Cells in Augmenting Autologous Fat Transfer, U.S. PatentPublication 2005/0025755, Yoshimura, et al., Characterization of FreshlyIsolated and Cultured Cells Derived form the Fatty and Fluid Portions ofliposuction Aspirates, J. Cell. Physiol. 208: 1011-1041 (2006);Yoshimura, et al., Cell-Assisted Lipotransfer for Facial Lipoatrophy:Effects of Clinical Use of Adipose-Derived Stem Cells, Dermatol. Surg.34: 1178-1185 (2008); Yoshimura, et al., Cell-Assisted Lipotransfer forCosmetic Breast Augmentation: Supportive Use of Adipose-DerivedStem/Stromal Cells, Aesth. Plast. Surg. 32: 48-55 (2008); each of whichis hereby incorporated by reference in its entirety.

In addition, harvested adipose tissue can be supplemented withregenerative cells obtained from cell cultures, such as, e.g., primarycell cultures and established cell cultures. For example, a portion ofharvested adipose tissue from an individual can be cultured in a mannerto produce primary cell cultures enriched for regenerative cells.Alternatively, established cell lines derived from regenerative cellsfrom adipose tissue, or another tissue source, can be cultured,harvested, and added to adipose tissue collected form an individual.Exemplary methods describing such cell culture compositions andprocedures can be found in, e.g., Casteilla, et al., Method forCulturing Cells Derived from the Adipose Tissue and Uses Thereof, U.S.Patent Publication 2009/0246182; Chazenbalk, et al, Methods of ProducingPreadipocytes and Increasing the Proliferation of Adult AdiposeStem/Progenitor Cells, U.S. Patent Publication 2009/0317367; Kleinsekand Soto, Augmentation and Repair of Sphincter Defects with CellsIncluding Adipocytic Cells, U.S. Patent Publication 2008/0299213;Rehman, et al., Secretion of Angiogenic and Antiapoptotic Factors byHuman Adipose Stromal Cells, Circulation 109: r52-r58 (2004); Kilroy, etal., Cytokine Profile of Human Adipose-Derived Stem Cells: Expression ofAngiogenic, Hematopoietic, and Pro-Inflammatory Factors, J. Cell.Physiol. 212: 702-709 (2007); each of which is hereby incorporated byreference in its entirety.

Harvested adipose tissue may be immediately used to make thecompositions disclosed herein. Alternatively, harvested adipose tissue,whether unprocessed or processed, may be stored for used at some futuredate. Harvested tissue is typically stored using a slow freezing methodof the tissue to −20° C., with or without cryopreservatives. Storedadipose tissue can typically be stored for at least 6 months.

In some embodiments, a hydrogel composition as described herein mayinclude a hyaluronic acid: silk fibroin weight ratio of 17 to 4. Theconcentrations of hyaluronic acid can be from about 2 mg/mL to about 25mg/mL and the silk fibroin can be from about 0.5 mg/mL to about 12mg/mL. Further, the hydrogel composition may be used for fat graftingapplications as an additive. The hydrogels can be formed with an EDCcrosslinker and NHS as an activating agent.

Hydrogel compositions described herein can further have a storagemodulus (G′) and a loss modulus (G″) each independently between about500 Pa and about 4,000 Pa.

A general method of making hydrogel compositions as described herein canbe achieved as follows. First, lyophilized hyaluronic acid fibers can beadded to a concentrated (e.g., hydrated) silk fibroin solution. The pHcan then be managed by the addition of one or more buffer salt and/orthe addition of a base (e.g., NaOH). After the pH has been managed, themixture can be hydrated and thoroughly mixed followed by addition ofcrosslinking agents. The crosslinking agents can be solids (e.g.,powder). The hyaluronic acid and silk fibroin can be left to react. Oncereacted, the resultant gel can be particle sized through a filter mesh(e.g., 100 μm) and can be dialyzed with buffer to purify (e.g., againstany unused or unreacted crosslinker). The gel can then be sterilized(e.g., using isopropanol). This sterilization can also occur prior topurification. Once sterilized the gel may be ready for administration.The sterilized gel can also be further mixed within adipose tissue(e.g., human).

The sterilized gel either mixed with adipose tissue or not mixed withadipose tissue can be administered as described herein to treat acondition of, for example, the face, breast, hands, etc.

EXAMPLES

The methods and compositions of the present disclosure are furtherdescribed in the following examples.

Example 1: Preparation of a Water-Soluble Silk Fibroin Solution

A 9.3 M LiBr solution was prepared by slowly dissolving 77.54 g of LiBrin 76.28 mL of MilliQ water. The LiBr solution was kept at 60° C. 24 gof sericin extracted knitted silkworm silk yarn was slowly submerged inthe LiBr solution. The LiBr and silk solution was incubated in an ovenat 60° C. for 6 hours. The solution was then loaded into a dialysiscassette MWCO 3.5 KDa and dialyzed against MilliQ water in a 4 L beakerat room temperature for 72 hours, changing the water at 1 hour, 4 hours,12 hours and then twice a day.

Example 2: 80% HA-20% Silk Fibroin Crosslinked Gel Made Using EDCChemistry and HMDA

1.2 mL of a 7 wt % Silk Fibroin (SF) MilliQ water solution and 20 mg ofthe diamine cross linker HMDA.2HCl was added to 13.8 mL of MilliQ water.336 mg of high molecular weight hyaluronic acid (HA) was added to thesolution. The mixture was allowed to hydrate for 60 minutes andhomogenized by passing 30 times syringe-to-syringe. 576 mg of MES bufferwas mixed with 321.6 mg of EDC and 72.9 mg of sulfoNHS in 5 mL MilliQwater. The reagent solution was mixed to the HA/SF solution by passingbetween syringes 30 times. The mixture was transferred to glass vialsand left to react overnight at 4° C. The gel was sized using a 100 ummesh and centrifuged at 4000 rpm for 5 minutes. The sized gel wastransferred to a cellulose ester membrane dialysis tubing MWCO 20 KDaand dialyzed against 1 X PBS for 8 days at room temperature, changingthe buffer twice a day. After dialysis, the gel was sized using a 60 ummesh, dispensed in 1 ml COC syringes, centrifuged at 5000 RPM for 5 min,and moist heat sterilized for 5 minutes at 128° C.

Example 3: 80% HA-20% SF Gel Cross Linked Using BDDE

2.4 mL of a 7 wt % Silk Fibroin (SF) MilliQ water solution and 1.25 mLof 1N NaOH MilliQ water solution were added to 1.35 mL MilliQ water. 494mg of HMW HA was added to the SF/NaOH solution and allowed to hydratefor 60 minutes and homogenized by passing 30 times syringe-to-syringe.85 mg of BDDE was added to the mixture and passed between syringes 30times. The mixture was cured in a water bath at 50° C. for 2 hours. Thegel was neutralized by adding 135 μL of 37% HCl and 4.86 mL PBS andpassed between syringes 30 times. 7.5 mL of PBS was added to the gel andthe gel was allowed to swell overnight at 4° C. The final HA/SFconcentration of the gel was about 5 wt %. The gel was sized using a 100um mesh and centrifuged at 4000 rpm for 5 minutes. The sized gel wastransferred to a cellulose ester membrane dialysis tubing MWCO 20 KDaand dialyzed against 1 X PBS for 8 days at 4° C., changing the buffertwice a day. After dialysis, the gel was dispensed in 1 ml COC syringes,centrifuged at 5000 RPM for 5 min, and sterilized with moist steam for 5minutes at 128° C.

Example 4: Synthesis of Hyaluronic Acid—B. Mori Silk Fibroin Hydrogelswith 5% Silk Fibroin Content

The following procedure was used to produce hydrogels with a 20:1 (5%)HA-silk fibroin composition. 24.4 mg lysine methyl ester (LME) wasdissolved in 14.54 mL MiIIiQ water. 0.46 mL of a 7% silk fibroin MiIIiQwater solution was dispersed in the solution. 393.3 mg hyaluronic acid,2 MDa molecular weight, was added and left to hydrate for 1 hour. Thesolution was homogenized by syringe-to-syringe mixing. 576.0 mg2-[morpholino] ethanesulfonic acid, 321.6 mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 72.9 mgN-hydroxysulfosuccinimide were added to 5 mL MiIIiQ water and mixed tothe hyaluronic acid/silk fibroin/lysine methyl ester solution bysyringe-to-syringe mixing. The solution was transferred to a glass vialand centrifuged for 5 min at 4000 RPM to remove air bubbles. Theresulting gel was allowed to react for 16 hrs at 4 aC. The gel was thenparticulated through a 60 micron pore-sized mesh. Following sizing, thegel was dialyzed in PBS pH7.4 for 6 days through a 20 kDamolecular-weight cut-off cellulose ester membrane at 4° C. The gel wasthen dispensed into 1 mL syringes, centrifuged at 4000 rpm for 10minutes to eliminate any non-absorbed water and sterilized byautoclaving in wet steam at 128° C. for 5 minutes.

Example 5: Synthesis of Hyaluronic Acid—B. Mori Silk Fibroin Hydrogelswith 20% Silk Fibroin Content

The following procedure was used to produce hydrogels with a 17:4 (20%)HA-silk fibroin composition. 24.4 mg lysine methyl ester (LME) wasdissolved in 13.8 mL MiIIiQ water. 336.0 mg hyaluronic acid, 2 MOamolecular weight, was added and left to hydrate for 1 hour. The solutionwas homogenized by syringe-to-syringe mixing. 1.2 mL of a 7% silkfibroin MilliQ water solution was mixed to the hyaluronic acid using astatic mixer. 576.0 mg 2-[morpholino] ethanesulfonic acid, 321.6 mg1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 72.9 mgN-hydroxysulfosuccinimide were added to 5 mL MiIIiQ water and mixed tothe hyaluronic acid/silk fibroin solution using the static mixer. Thesolution was transferred to a glass vial and centrifuged for 5 min at4000 RPM to remove air bubbles. The resulting gel was allowed to reactfor 16 hrs at 4° C. The gel was then particulated through a 60 micronpore-sized mesh. Following sizing, the gel was dialyzed in PBS pH7.4 for6 days through a 20 kOa molecular-weight cut-off cellulose estermembrane at 4° C. The gel was then dispensed into 1 mL syringes,centrifuged at 4000 rpm for 10 minutes to eliminate any non-absorbedwater and sterilized by autoclaving in wet steam at 128° C. for 5minutes.

Example 6: Rheological Characterization of Hyaluronic Acid—B. Mori SilkFibroin Hydrogels

Oscillatory parallel plate rheology was used to characterize themechanical properties of the hydrogels synthesized in Example 4 andExample 5 using an Anton Paar MCR 301. A plate diameter of 25 mm wasused at a gap height of 1 mm. A frequency sweep from 0.1 to 10 Hz at afixed strain of 2% with logarithmic increase in frequency was appliedfollowed by a strain sweep between 0.1% and 300% at a fixed frequency of5 Hz with logarithmic increase in strain. The storage modulus (G′) andloss modulus (G″) were recorded from frequency sweep measurements at 5Hz. Values from measurements of samples from Examples 4 and 5 arepresented in Table 1.

TABLE 1 Rheological properties of hyaluronic acid-silk fibroin (HA-Fbn)hydrogels [HA] [Fbn] G′ G″ Sample ID (mg/mL) (mg/mL) (Pa) (Pa) HA-Fbn20:1 20 1 1170 31.7 HA-Fbn 17:4 17 4 1250 47.3 HA-Fbn 17:4 (A) 17 4 103040.0 HA-Fbn 17:4 (B) 17 4 867 31.6 HA-Fbn 17:4 (C) 17 4 1200 53.7 HA-Fbn17:4 (D) 17 4 1010 41.9

Example 7: Swelling Ratios of Hyaluronic Acid—B. Mori Silk FibroinHydrogels

Swelling ratios were determined using thermogravimetric analysis (TGA)on HA-silk fibroin (A), (B) and (D) hydrogels synthesized in Example 5.The solid content variation in the gels was calculated afterequilibration with phosphate buffer. For each gel, approximately 1 mLwas dispensed into a 15 mL Falcon tube, followed by addition of 10 mL ofphosphate buffered saline, pH 7.4. The gels were thoroughly mixed withthe buffer and vortexed for 30 seconds. The gels were then allowed toequilibrate in the buffer for 24 hrs at room temperature. After thistime, the suspensions were centrifuged at 4000 RPM in a swinging bucketrotor for 5 minutes. The supernatant buffer was then decanted and thegels were dispensed into 1 mL syringes, centrifuged at 4000 rpm for 10minutes to eliminate any non-absorbed water. The TGA measurements wereperformed in triplicates. The swelling ratio was determined by dividingthe final solid content of the swollen gel by the solid content feed,i.e., hyaluronic acid, silk fibroin and crosslinker. The swellingresults of samples from Example 5 are presented in Table 2. A swellingratio less than 1 indicates that the gel lost water upon equilibrationand centrifugation.

TABLE 2 Swelling ratios of hyaluronic acid-silk fibroin hydrogelsAverage [HA] [Fbn] Swelling Sample ID (mg/mL) (mg/mL) Ratio HA-Fbn 17:4(A) 17 4 1.38 HA-Fbn 17:4 (B) 17 4 1.59 HA-Fbn 17:4 (D) 17 4 1.83

Example 8: Biocompatibility of Hyaluronic Acid—B Mori Silk FibroinHydrogels

Biocompatibility was tested on HA-silk fibroin 20:1 (Example 4) andHA-silk fibroin 17:4 (Example 5). Results were compared to Juvederm™ XP(Allergan), an injectable dermal filler comprised of a cross-linkedhyaluronic acid, and Star-HA hydrogel, which contain only hyaluronicacid and multi-epoxy crosslinkers. These gels are known to cause low tomoderate inflammation in vivo. Male 14-weeks old Sprague-Dawley ratswere administered 50 μL of hydrogel using a 27 gauge needle. Injectionswere placed intradermally on the dorsum of each animal under anesthesiawith four injections per rat. After one week, the gels were explanted.Sections were cut around each implant in its entirety, including thesurrounding tissue defined by a radius of approximately 1 cm. CD68staining assay to assess the degree of CD68 macrophage marker wereperformed on the explanted gels for histology. CD68 scores weredetermined for each of the gels, and are presented in Table 3 below. Allaverage scores were below 3, which indicates low to moderateinflammation. The results obtained for HA-silk fibroin hydrogels werestatistically identical to Star HA. Thus, the HA-silk fibroin hydrogelsshow low levels of inflammation, indicating biocompatibility.

TABLE 3 Bicompatibilitv CD68 scores of hyaluronic acid-silk fibroin(HA-Fbn) hydrogels [HA] [Fbn] Sample Crosslinker (mg/mL) (mg/mL) AverageScore Juvederm XP BDDE 24 0 0.83 ± 0.61 Star HA PEG-epoxide 26 0 2.50 ±1.10 HA-Fbn 20:1 LME 20 1 1.92 ± 0.63 HA-Fbn 17:4 LME 17 4 2.99 ± 0.74

Example 9: Attachment of Human Adipose Derived Stem Cell (hASC) onHyaluronic Acid—B. Mori Silk Fibroin Hydrogels

The HA-silk fibroin 20:1 (Example 4) and 17:4 (Example 5) hydrogels weretested for hASC attachment. 100 μL gel beds were made in a non-TCP(tissue culture polystyrene) 48-well plate and hASC having aconcentration of 6×10⁴ hASC/mL were placed on top of the gel beds. Cellswere cultured overnight at 37° C. The next day, cells were stained withCalcein AM for 30 min. and viewed with an inverted fluorescentmicroscope. Microscope focus was set on the bottom of the wells and wasmoved up to locate the attached cells. The results were compared to hASCattachment to a TPC plate. An examination of the micrographs showed thecells attached to both samples, but showed a significant spreading(rather than concentrated) on the HA-Fbn 17:4 hydrogel, indicating thepositive effect of fibroin on cell attachment to the hydrogel, as wellas the effect of the concentration of fibroin in the hydrogel on cellattachment.

Example 10: Support of hASC Viability on Hyaluronic Acid—B. Mori SilkFibroin Hydrogels

Samples of HA-Fbn hydrogels 17:4 from Example 5 were tested for theirability to support human adipose derived stem cell (hASC) viability. In96-well plates, 50 μL gel beds were created in triplicate from thehydrogels of Example 5. Culture-expanded ASCs (Invitrogen) were platedat 10,000 cells/well on the gel beds in MesenPro RS medium with growthsupplement (Invitrogen, CA). The cells were cultured for 18 hrs at 37°C., 5% CO₂, after which the XTT assay (American Type Culture Collection,VA) was performed. The2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbonxanilidesalt (XTT) is added to the wells and the cells are incubated. XTT is acolorless compound and when reduced by metabolically active cells itbecomes a bright orange substance. The formazan product of XTT reductionis soluble so the absorbance can be read using a spectrophotometer at475 nm and at 660 nm for non-specific readings. Cells adhered to thegels and exhibited a spread morphology. The viability of hASCs increasedwith increasing total biopolymer and collagen concentrations. Viabilityrelative to tissue culture polystyrene (TCP) are shown in Table 4.Results are statistically similar, although the average result isslightly higher for some of the HA-Fbn hydrogels.

TABLE 4 ASC viability on hyaluronic acid-silk fibroin (HA-Fbn) hydrogels[HA] [Fbn] Viability Sample (mg/mL) (mg/mL) (relative to TCP) HA-Fbn17:4 (A) 17 4 52% HA-Fbn 17:4 (B) 17 4 55% HA-Fbn 17:4 (C) 17 4 81%HA-Fbn 17:4 (D) 17 4 57%

Example 11: Support of hASC Proliferation on Hyaluronic Acid—B. MoriSilk Fibroin (HA-Fbn) Hydrogels

Samples of HA-Fbn from Example 5 were tested for their ability tosupport ASC proliferation. In 96-well plates, 50 μL gel beds werecreated in triplicate from the HA-Fbn hydrogels. The hASCs (Invitrogen)were plated at 6,000 cells/well on the gel beds in MesenPro RS mediumwith growth supplement (Invitrogen). The hASCs were cultured for 3 daysat 37° C., 5% CO₂, and proliferation was determined by XTT assay.Proliferation rates at day 3, relative to TCP. Results are shown inTable 5 and are statistically similar, although the average result islightly higher for some of the HA-Fbn hydrogels.

TABLE 5 hASC proliferation (3-day) on hyaluronic acid-silk fibroin(HA-Fbn) hydrogels [HA] [Fbn] Proliferation (%) Sample (mg/mL) (mg/mL)(relative to TCP) HA-Fbn 17:4 (A) 17 4 29 HA-Fbn 17:4 (B) 17 4 46 HA-Fbn17:4 (C) 17 4 74 HA-Fbn 17:4 (D) 17 4 42

Example 12: Enhanced Diffusion of Adipose Tissue-Specific andPro-Angiogenic Growth Factors in Hyaluronic Acid—B. Mori Silk FibroinHydrogels

Two batches, HA-silk fibroin 17:4 (A) and (C) from Example 5 wereassessed for their ability to allow diffusion of pro-angiogenic(vascular endothelial growth factor, VEGF) and adipose tissue-specificgrowth factors (adiponectin, leptin). Improved diffusion to any or allof these growth factors would support the enhanced survival ofco-grafted tissue, especially fat, since nutrient diffusion may beimportant for sustained tissue viability. To do this, 100 μL of eachhydrogel tested was loaded into a 8 μm transwell (24 well plate) inorder to make a gel column. Known concentrations of target factors wereloaded on top of the gel, diluted in fibroblast basal medium(Cat#PCS-201-030, ATCC). Plates were allowed to incubate at 37° C. with5% CO₂ in a tissue culture incubator, for 60 hours, thereby allowing thefactors to diffuse through the gels. Diffusion of the specified factorsthrough each hydrogel was measured by ELISA (enzyme-linked immunosorbentassay) in the supernatant in the bottom chamber of the wells. Resultsare shown in Table 6 and indicate improved diffusion of VEGF andAdiponectin over the reference, Matrigel™ (Corning Life Sciences).

TABLE 6 Diffusion of growth factors and adipokines in hyaluronicacid-silk fibroin (HA-Fbn) hydrogels Sample Adiponectin (%) Leptin (%)VEGF (%) Matrigel 19 44 30 HA-Fbn 17:4 (A) 82 18 79 HA-Fbn 17:4 (B) 7418 71

Example 13: Enhanced Diffusion of Sugar Polymers and Proteins inHyaluronic Acid—B. Mori Silk Fibroin (HA-Fbn) Hydrogels

Hyaluronic acid silk fibroin (A) and (C) from Example 5 were assessedfor their ability to allow diffusion of sugar polymers and proteins.Improved diffusion to any or all of these substances would support theenhanced survival of co-grafted tissue, especially fat, since nutrientdiffusion is a critical element to sustained tissue viability. To dothis, 100 μL of each hydrogel tested was loaded into a 8 μm transwell(24 well plate) in order to make a gel column. Known concentrations oftarget factors were loaded on top of the gel, diluted in fibroblastbasal medium (Cat#PCS-201-030, ATCC). Plates were allowed to incubate at37° C. with 5% CO₂ in a tissue culture incubator, for 60 hours, therebyallowing the factors to diffuse through the gels. Diffusion of thespecified factors through each hydrogel was measured by ELISA using thesupernatant in the bottom chamber of the wells. Diffusion was determinedrelative to no gel present. Results are shown in Table 7 and indicateimproved levels of diffusion for FITC-dextran and total protein,compared to the positive control, Matrigel™.

TABLE 7 Diffusion of sugar polymers and proteins in hyaluronic acid-silkfibroin (HA-Fbn) hydrogels Sample FITC-Dextran (%) Total Protein (%)Matrigel 32 38 HA-Fbn 17:4 (A) 72 91 HA-Fbn 17:4 (B) 71 93

Example 14: Enhanced 3D Adipogenesis on Hyaluronic Acid—B. Mori SilkFibroin (HA-Fbn) Hydrogels

Hyaluronic acid-silk fibroin (A) from Example 5 was assessed for itsability to allow differentiation of hASCs to adipocytes. The physicaland biological properties of the gel contribute to cell attachment,migration, and cell-cell and cell-matrix interactions, which dictate thedifferentiation of hASCs To evaluate the combined effects of thesefactors, a 3D culture was used for assessing adipogenesis capacity ofHA-Fbn hydrogels. To do this, 500 μL of the hydrogel was loaded in to a0.4 μm transwell (24 well plate) in order to make a gel column. ThehASCs (Invitrogen) in 100 μL cell solution were plated at 1 millioncells/well on the gel beds in Adipogenesis Media with AdipogenesisDifferentation Supplement (Invitrogen). Plates were allowed to incubateat 37° C. with 5% CO₂ in tissue culture incubator for 3 days, at whichpoint the media was changed. At days 7, 14, 21, 28, and 35, media wascollected and measured by ELISA using the supernatant in the bottomchamber of the wells. The secretion of adipose tissue-specific growthfactors, such as diponectin, was assessed as an indication of adipocytedifferentiation. The results are shown in Table 8 and show improvedlevels of adiponectin for HA-Fbn compared to the positive controlHA-Collagen 12:6 (HA-CN 12:6).

TABLE 8 Adiponectin levels from differentiated adipocytes on hyaluronicacid-silk fibroin hydrogel Adiponectin Adiponectin Adiponectin [ng/mL][ng/mL] [ng/mL] Sample Day 21 Day 28 Day 35 HA-CN 12:6 13 35 25 HA-Fbn17:4 (A) 35 42 58

Example 15: In Vivo Volume Retention Studies of Hyaluronic Acid-SilkFibroin (HA-Fbn) Hydrogels

Three HA-Fbn hydrogels were prepared in a similar manner described tothat of Examples 4 and 5 with HA:Fbn ratios of 19:2, 18:3, and 17:4.These hydrogels were mixed with human lipoaspirate at 2:1 lipo:gel ratioin a nude mouse model to assess the gels' ability to enhance fat graftviability and volume retention. Human lipoaspirate tissue was procuredthrough means of ultrasound- or suction-assisted liposuction underinformed consent, then consecutively centrifuged and washed 3× at 30 gfor 5 min, in 1× phosphate buffered saline without cations (PBS,Invitrogen) inside a sterile biosafety cabinet. Next, 10 mL of washedlipoaspirate was transferred to a clean 100 mL sterile reservoir. Tothis tissue, 5 mL of sterile hydrogel was added and carefully blended byhand using a sterile spatula. The mixing procedure required 5 to 10minutes of constant stirring with mechanical disruption of large piecesof tissue to generate a homogenous mixture. Then 1 mL syringes werefilled with lipoaspirate/hydrogel until the plunger reached the 1 mLmark. The syringe was then capped with a sterile female leur-lok cap andmaintained on ice blocks until use. Lipoaspirate/hydrogel mixes wereimplanted as 1 mL bolus subcutaneously on the dorsum of female6-week-old nude mice under anesthesia with two injections per mouse.Each gel/lipo mixture was implanted through a small incision by 16 Gcannula and the incision closed using surgical glue. A total of 14injections of each material were made. Syringes were weighed before andafter injection to determine the weight of injected material. After 6weeks, the gels were harvested and weight and volume (using liquiddisplacement) were determined for each sample. A Lipo-only controlsample was also tested. Samples were processed for histology by H&Estaining.

FIG. 1 provides a graph of the volume retention over time of the threesamples in comparison to the Lipo-only control. All of the tested HA-Fbnhydrogels have significantly higher volume than the Lipo-only controlafter 6 weeks. The HA-Fbn 17:4 shows significantly higher volume thanthe control. The volume retention for each of the samples and that ofthe Lipo-only control are provided in Table 9 below. It can be seen thatthe volume retention improved significantly over the Lipo-only control.

TABLE 9 Volume retention after 6 weeks for hyaluronic acid-silk fibroinhydrogel (HA-Fbn) fat grafts compared to a lipoaspirate-only controlAverage Retained Improvement over Lipo-only Sample Volume (%) control(%) Lipo only 68.23 ± 9.63  N/A Lipo + HA-Fbn 19:2 90.57 ± 14.63 22.34Lipo + HA-Fbn 18:3 90.29 ± 18.06 22.06 Lipo + HA-Fbn 17:4 90.93 ± 10.5622.70

FIG. 2A exhibits a photograph of the harvested Lipo-only control gel,and FIG. 3B provides a micrograph at 5× magnification of the H&E stainedhistological samples of the harvested Lipo-only control. The extractedLipo-only control sample showed a lower amount of angiogenesis asindicated by the scant presence of darkened tissue in the photograph ofFIG. 2A. Likewise, the micrograph of the Lipo-only sample shown in FIG.2B shows little cell growth and angiogenesis. In this micrograph, it canbe seen that most of the adipocytes in the Lipo-only sample are dead.

In contrast, the photographs of the harvested HA-Fbn samples 19:2 (FIG.3A), 18:3 (FIG. 4A), and 17:4 (FIG. 5A) all show an increase of darkenedtissue areas, indicating a greater amount of angiogenesis than theLipo-only control. In addition, the micrographs at 5× magnification ofthe H&E stained histological samples of the harvested HA-Fbn samples19:2 (FIG. 3B), 18:3 (FIG. 4B), and 17:4 (FIG. 5B) show that theseextracted samples contain more viable adipocytes than the Lipo-onlycontrol. Additionally, these samples all exhibit good cell infiltrationand good angiogenesis, with low to moderate inflammation.

Illustration of Subject Technology as Clauses

Various examples of aspects of the disclosure are described as numberedclauses (1, 2, 3, etc.) for convenience. These are provided as examples,and do not limit the subject technology. Identifications of the figuresand reference numbers are provided below merely as examples and forillustrative purposes, and the clauses are not limited by thoseidentifications.

Clause 1. A soft tissue augmentation product comprising: a formingcomponent comprising a hydrogel composition; wherein the hydrogelcomposition comprises water and a crosslinked macromolecular matrix, thecrosslinked macromolecular matrix comprising: a hyaluronic acidcomponent; and a silk fibroin component; wherein the hyaluronic acidcomponent is crosslinked to the silk fibroin component by a multiaminecross linker; and wherein the soft tissue augmentation product isconfigured for administration to a soft tissue of a human subject.

Clause 2. The product of Clause 1, wherein the forming componentcomprises an injectable composition.

Clause 3. The product of any one of the preceding Clauses, wherein theforming component is configured for implantation into a human softtissue.

Clause 4. The product of any one of the preceding Clauses, wherein theforming component further comprises human adipose tissue.

Clause 5. The product of Clause 4, wherein the human adipose tissue isautologous with the soft tissue.

Clause 6. The product of Clause 5, wherein the human adipose tissuecomprises a lipoaspirate.

Clause 7. The product of any one of the preceding Clauses, furthercomprising a label comprising instructions to administer the formingcomponent into a soft tissue.

Clause 8. The product of any one of the preceding Clauses, wherein themultiamine cross linker comprises a diamine cross linker.

Clause 9. The product of Clause 8, wherein the multiamine cross linkeris selected from the group consisting of a hexamethylene diamine (HMDA),lysine, lysine methyl ester, and lysine ethyl ester.

Clause 10. The product of Clause 9, wherein the multiamine cross linkeris lysine methyl ester.

Clause 11. The product of any one of the preceding Clauses, wherein thesilk fibroin component comprises a B. mori silk fibroin.

Clause 12. The product of any one of the preceding Clauses, wherein thecrosslinked macromolecular matrix has a weight ratio of the hyaluronicacid component to the silk fibroin component in the range of about 25:1to about 1:1.

Clause 13. The product of Clause 12, wherein the crosslinkedmacromolecular matrix has a weight ratio of the hyaluronic acidcomponent to the silk fibroin component of about 20:1.

Clause 14. The product of Clause 12, wherein the crosslinkedmacromolecular matrix has a weight ratio of the hyaluronic acidcomponent to the silk fibroin component of about 17:4.

Clause 15. The product of Clause 12, wherein the crosslinkedmacromolecular matrix has a weight ratio of the hyaluronic acidcomponent to the silk fibroin component of about 18:3.

Clause 16. The product of any one of the preceding Clauses, wherein thehyaluronic acid component is present in the hydrogel composition in aconcentration of about 20 mg/mL to about 40 mg/mL, and wherein the silkfibroin component is present in the hydrogel composition in aconcentration of about 0.1 mg/mL to about 20 mg/mL.

Clause 17. The product of Clause 16, wherein the hyaluronic acidcomponent is present in the hydrogel composition in a concentration ofabout 9 mg/mL to about 32 mg/mL, and wherein the silk fibroin componentis present in the hydrogel composition in a concentration of about 1mg/mL to about 8 mg/mL.

Clause 18. The product of Clause 16, wherein the hyaluronic acidcomponent is present in the hydrogel composition in a concentration ofabout 16 mg/mL to about 20 mg/mL, and wherein the silk fibroin componentis present in the hydrogel composition in a concentration of about 2mg/mL to about 5 mg/mL.

Clause 19. The product of Clause 16, wherein the hyaluronic acidcomponent is present in the hydrogel composition in a concentration ofabout 17 mg/mL, and wherein the silk fibroin component is present in thehydrogel composition in a concentration of about 4 mg/mL.

Clause 20. The product of Clause 16, wherein the hyaluronic acidcomponent is present in the hydrogel composition in a concentration ofabout 18 mg/mL, and wherein the silk fibroin component is present in thehydrogel composition in a concentration of about 3 mg/mL.

Clause 21. The product of Clause 16, wherein the hyaluronic acidcomponent is present in the hydrogel composition in a concentration ofabout 19 mg/mL, and wherein the silk fibroin component is present in thehydrogel composition in a concentration of about 2 mg/mL.

Clause 22. The product of any one of the preceding Clauses, wherein thehyaluronic acid component has a molecular weight of about 1,000,000daltons to about 5,000,000 daltons.

Clause 23. The product of any one of the preceding Clauses, wherein thehyaluronic acid component has a molecular weight of about 1,000,000daltons to about 3,000,000 daltons.

Clause 24. A method of augmenting soft tissue of a human beingcomprising: providing a hydrogel composition comprising water and acrosslinked macromolecular matrix, the crosslinked macromolecular matrixcomprising a hyaluronic acid component and a silk fibroin component,wherein the hyaluronic acid component is crosslinked to the silk fibroincomponent by a multiamine cross linker; and mixing the hydrogelcomposition with an ex vivo adipose tissue to produce a hydrogel-adiposetissue mixture.

Clause 25. The method of Clause 24, further comprising the step ofintroducing the hydrogel-adipose tissue mixture into a soft tissue ofthe human being.

Clause 26. The method of Clause 25, wherein the step of introducingcomprises injecting the hydrogel-adipose tissue mixture into a softtissue of the human being.

Clause 27. The method of Clause 25, wherein the step of introducingcomprises configuring the hydrogel-adipose tissue mixture forimplantation into a soft tissue of a human being and implanting theconfigured hydrogel-adipose tissue mixture into the soft tissue of thehuman being.

Clause 28. The method of any one of Clauses 24 to 27, wherein theadipose tissue comprises cells, the cells including adipocytes, multipletypes of regenerative cells, stromal vascular fraction cells, or acombination thereof.

Clause 29. The method of any one of Clauses 24 to 28, wherein theadipose tissue is autologous with the soft tissue of the human being.

Clause 30. The method of Clause 29, wherein the adipose tissue comprisesa lipoaspirate.

Clause 31. The method of any one of Clauses 24 to 30, wherein themultiamine cross linker comprises a diamine cross linker.

Clause 32. The method of Clause 31, wherein the multiamine cross linkeris selected from the group consisting of a hexamethylene diamine (HMDA),lysine, lysine methyl ester, and lysine ethyl ester.

Clause 33. The method of Clause 32, wherein the multiamine cross linkercomprises lysine methyl ester.

Clause 34 The method of any one of Clauses 24 to 33, wherein the silkfibroin component comprises a B. mori silk fibroin.

Clause 35. The method of any one of Clauses 24 to 34, wherein thecrosslinked macromolecular matrix has a weight ratio of the hyaluronicacid component to the silk fibroin component in the range of about 25:1to about 1:1.

Clause 36. The method of Clause 35, wherein the crosslinkedmacromolecular matrix has a weight ratio of the hyaluronic acidcomponent to the silk fibroin component of about 20:1.

Clause 37. The method of Clause 35, wherein the crosslinkedmacromolecular matrix has a weight ratio of the hyaluronic acidcomponent to the silk fibroin component of about 17:4.

Clause 38. The method of Clause 35, wherein the crosslinkedmacromolecular matrix has a weight ratio of the hyaluronic acidcomponent to the silk fibroin component of about 18:3.

Clause 39. The method of any one of Clauses 24 to 38, wherein thehyaluronic acid component is present in the hydrogel composition in aconcentration of about 20 mg/mL to about 40 mg/mL, and wherein the silkfibroin component is present in the hydrogel composition in aconcentration of about 0.1 mg/mL to about 20 mg/mL.

Clause 40. The method of Clause 39, wherein the hyaluronic acidcomponent is present in the hydrogel composition in a concentration ofabout 9 mg/mL to about 32 mg/mL, and wherein the silk fibroin componentis present in the hydrogel composition in a concentration of about 1mg/mL to about 8 mg/mL.

Clause 41. The method of Clause 39, wherein the hyaluronic acidcomponent is present in the hydrogel composition in a concentration ofabout 16 mg/mL to about 20 mg/mL, and wherein the silk fibroin componentis present in the hydrogel composition in a concentration of about 2mg/mL to about 5 mg/mL.

Clause 42. The method of Clause 39, wherein the hyaluronic acidcomponent is present in the hydrogel composition in a concentration ofabout 17 mg/mL, and wherein the silk fibroin component is present in thehydrogel composition in a concentration of about 4 mg/mL.

Clause 43. The method of Clause 39, wherein the hyaluronic acidcomponent is present in the hydrogel composition in a concentration ofabout 18 mg/mL, and wherein the silk fibroin component is present in thehydrogel composition in a concentration of about 3 mg/mL.

Clause 44. The method of Clause 39, wherein the hyaluronic acidcomponent is present in the hydrogel composition in a concentration ofabout 19 mg/mL, and wherein the silk fibroin component is present in thehydrogel composition in a concentration of about 2 mg/mL.

Clause 45. The method of any one of Clauses 24 to 44, wherein thehyaluronic acid component has a molecular weight of about 1,000,000daltons to about 5,000,000 daltons.

Clause 46. The method of any one of Clauses 24 to 44, wherein thehyaluronic acid component has a molecular weight of about 1,000,000daltons to about 3,000,000 daltons.

Clause 47. A method of grafting fat in a human subject, the methodcomprising providing a composition, wherein the composition comprises:(i) a hydrogel comprising: water and a crosslinked macromolecularmatrix, the crosslinked macromolecular matrix comprising a hyaluronicacid component and a silk fibroin component, wherein the hyaluronic acidcomponent is crosslinked to the silk fibroin component by a multiaminecross linker; and (ii) a fat component, comprising adipose tissue,adipocytes, or both.

Clause 48. The method of Clause 47, wherein the fat component has beenexplanted from the human subject.

Clause 49. The method of Clause 48, wherein the fat component comprisesa lipoaspirate.

Clause 50. The method of any one of Clauses 47 to 49, further comprisingthe step of administering the composition to soft tissue of the humansubject, thereby increasing the volume of fat in the soft tissue of thesubject.

Clause 51. The method of Clause 50, wherein the step of administeringthe composition results in an increase in fat graft volume retention ascompared to administering the fat component alone.

Clause 52. The method of any one of Clauses 50 to 51, wherein theadministering comprises injecting or implanting the composition into thesoft tissue of the human subject.

Clause 53. The method of any one of Clauses 47 to 52, wherein thecomposition has a fat component:hydrogel weight ratio of about 1:1 toabout 5:1.

Clause 54. The method of any one of Clauses 47 to 53, wherein themultiamine cross linker comprises a diamine cross linker.

Clause 55. The method of Clause 54, wherein the multiamine cross linkeris selected from the group consisting of a hexamethylene diamine (HMDA),lysine, lysine methyl ester, and lysine ethyl ester.

Clause 56. The method of Clause 55, wherein the multiamine cross linkeris lysine methyl ester.

Clause 57. The method of any one of Clauses 47 to 56, wherein the silkfibroin is a B. mori silk fibroin.

Clause 58. The method of any one of Clauses 47 to 57, wherein thecrosslinked macromolecular matrix has a weight ratio of hyaluronic acidto silk fibroin in the range of about 25:1 to about 1:1.

Clause 59. The method of Clause 58, wherein the crosslinkedmacromolecular matrix has a weight ratio of hyaluronic acid to silkfibroin of about 20:1.

Clause 60. The method of Clause 58, wherein the crosslinkedmacromolecular matrix has a weight ratio of hyaluronic acid to silkfibroin of about 17:4.

Clause 61. The method of Clause 58, wherein the crosslinkedmacromolecular matrix has a weight ratio of hyaluronic acid to silkfibroin of about 18:3.

Clause 62. The method of any one of Clauses 47 to 61, wherein thehyaluronic acid component is present in the hydrogel in a concentrationof about 20 mg/mL to about 40 mg/mL, and wherein the silk fibroincomponent is present in the hydrogel in a concentration of about 0.1mg/mL to about 20 mg/mL.

Clause 63. The method of Clause 62, wherein the hyaluronic acidcomponent is present in the hydrogel in a concentration of about 9 mg/mLto about 32 mg/mL, and wherein the silk fibroin component is present inthe hydrogel in a concentration of about 1 mg/mL to about 8 mg/mL.

Clause 64. The method of Clause 62, wherein the hyaluronic acidcomponent is present in the hydrogel in a concentration of about 16mg/mL to about 20 mg/mL, and the silk fibroin component is present inthe hydrogel in a concentration of about 2 mg/mL to about 5 mg/mL.

Clause 65. The method of Clause 62, wherein the hyaluronic acidcomponent is present in the hydrogel in a concentration of about 17mg/mL, and wherein the silk fibroin component is present in the hydrogelin a concentration of about 4 mg/mL.

Clause 66. The method of Clause 62, wherein the hyaluronic acidcomponent is present in the hydrogel in a concentration of about 18mg/mL, and wherein the silk fibroin component is present in the hydrogelin a concentration of about 3 mg/mL.

Clause 67. The method of Clause 62, wherein the hyaluronic acidcomponent is present in the hydrogel in a concentration of about 19mg/mL, and wherein the silk fibroin component is present in the hydrogelin a concentration of about 2 mg/mL.

Clause 68. The method of any one of Clauses 47 to 67, wherein thehyaluronic acid component has a molecular weight of about 1,000,000daltons to about 5,000,000 daltons.

Clause 69. The method of any one of Clauses 47 to 68, wherein thehyaluronic acid component has a molecular weight of about 1,000,000daltons to about 3,000,000 daltons.

Clause 70. The method of any one of Clauses 47 to 69, wherein the fatcomponent contains adipocytes, and wherein administering the compositionenhances adipocyte proliferation as compared to administering adipocytesalone.

Clause 71. The method of any one of Clauses 47 to 69, wherein the fatcomponent contains adipose tissue, and wherein administering thecomposition enhances adipose tissue growth as compared to administeringadipose tissue alone.

Clause 72. A method of grafting fat in a soft tissue of a human subject,the method comprising: (i) injecting a hydrogel component into the softtissue of the subject, wherein the hydrogel component comprises waterand a crosslinked macromolecular matrix, the crosslinked macromolecularmatrix comprising a hyaluronic acid component and a silk fibroincomponent, wherein the hyaluronic acid component is crosslinked to thesilk fibroin component by a multiamine cross linker; and (ii)administering a fat component to the soft tissue of the subject, whereinthe fat component contains adipose tissue, adipocytes, or both, andwherein the fat component has been explanted from the human subject;thereby increasing the volume of fat in the soft tissue of the humansubject.

Clause 73. The method of Clause 72, wherein the injection of thehydrogel component and the administration of the fat component to thesoft tissue is performed sequentially.

Clause 74. The method of Clause 73, wherein the injection of thehydrogel component to the soft tissue precedes the administration of thefat component to the soft tissue.

Clause 75. The method of any one of Clauses 72 to 74, wherein the fatcomponent is injected into the soft tissue.

Clause 76. The method of Clause 72, wherein the hydrogel component iscontacted with the fat component prior to the injection to provide asingle composition, which is injected into the soft tissue of the humansubject.

Clause 77. The method of Clause 76, wherein the composition has a fatcomponent:hydrogel weight ratio of 1:1 to 5:1.

Clause 78. The method of any one of Clauses 72 to 77, wherein themultiamine cross linker comprises a diamine cross linker.

Clause 79. The method of Clause 78, wherein the multiamine cross linkeris selected from the group consisting of a hexamethylene diamine (HMDA),lysine, lysine methyl ester, and lysine ethyl ester.

Clause 80. The method of Clause 79, wherein the multiamine cross linkeris lysine methyl ester.

Clause 81. The method of any one of Clauses 72 to 81, wherein the silkfibroin is a B. mori silk fibroin.

Clause 82. The method of any one of Clauses 72 to 81, wherein thecrosslinked macromolecular matrix has a weight ratio of the hyaluronicacid to the silk fibroin in the range of about 25:1 to about 1:1.

Clause 83. The method of Clause 82, wherein the crosslinkedmacromolecular matrix has a weight ratio of the hyaluronic acid to thesilk fibroin of about 20:1.

Clause 84. The method of Clause 82, wherein the crosslinkedmacromolecular matrix has a weight ratio of the hyaluronic acid to thesilk fibroin of about 17:4.

Clause 85. The method of Clause 82, wherein the crosslinkedmacromolecular matrix has a weight ratio of the hyaluronic acid to thesilk fibroin of about 18:3.

Clause 86. The method of any one of Clauses 72 to 85, wherein thehyaluronic acid component is present in the hydrogel component in aconcentration of about 20 mg/mL to about 40 mg/mL, and wherein the silkfibroin component is present in the hydrogel component in aconcentration of about 0.1 mg/mL to about 20 mg/mL.

Clause 87. The method of Clause 86, wherein the hyaluronic acidcomponent is present in the hydrogel component in a concentration ofabout 9 mg/mL to about 32 mg/mL, and wherein the silk fibroin componentis present in the hydrogel component in a concentration of about 1 mg/mLto about 8 mg/mL.

Clause 88. The method of Clause 86, wherein the hyaluronic acidcomponent is present in the hydrogel component in a concentration ofabout 16 mg/mL to about 20 mg/mL, and wherein the silk fibroin componentis present in the hydrogel component in a concentration of about 2 mg/mLto about 5 mg/mL.

Clause 89. The method of Clause 86, wherein the hyaluronic acidcomponent is present in the hydrogel component in a concentration ofabout 17 mg/mL, and wherein the silk fibroin component is present in thehydrogel component in a concentration of about 4 mg/mL.

Clause 90. The method of Clause 86, wherein the hyaluronic acidcomponent is present in the hydrogel component in a concentration ofabout 18 mg/mL, and wherein the silk fibroin component is present in thehydrogel component in a concentration of about 3 mg/mL.

Clause 91. The method of Clause 86, wherein the hyaluronic acidcomponent is present in the hydrogel component in a concentration ofabout 19 mg/mL, and wherein the silk fibroin is present in the hydrogelcomponent in a concentration of about 2 mg/mL.

Clause 92. The method of any one of Clauses 72 to 91, wherein thehyaluronic acid component has a molecular weight of about 1,000,000daltons to about 5,000,000 daltons.

Clause 93. The method of any one of Clauses 72 to 91, wherein thehyaluronic acid component has a molecular weight of about 1,000,000daltons to about 3,000,000 daltons.

Clause 94. The method of any one of Clauses 72 to 93, wherein fat graftvolume retention is increased as compared to administering the fatcomponent alone.

Clause 95. The method of any one of Clauses 72 to 94, wherein adipocyteproliferation is enhanced as compared to administering adipocytes alone.

Clause 96. The method of any one of Clauses 72 to 95, wherein adiposetissue growth is enhanced as compared to administering adipose tissuealone.

Further Considerations

In some embodiments, any of the clauses herein may depend from any oneof the independent clauses or any one of the dependent clauses. In someembodiments, any of the clauses (e.g., dependent or independent clauses)may be combined with any other one or more clauses (e.g., dependent orindependent clauses). In some embodiments, a Clause may include some orall of the words (e.g., steps, operations, means or components) recitedin a clause, a sentence, a phrase or a paragraph. In some embodiments, aClause may include some or all of the words recited in one or moreclauses, sentences, phrases or paragraphs. In some embodiments, some ofthe words in each of the clauses, sentences, phrases or paragraphs maybe removed. In some embodiments, additional words or elements may beadded to a clause, a sentence, a phrase or a paragraph. In someembodiments, the subject technology may be implemented without utilizingsome of the components, elements, functions or operations describedherein. In some embodiments, the subject technology may be implementedutilizing additional components, elements, functions or operations.

The foregoing description is provided to enable a person skilled in theart to practice the various configurations described herein. While thesubject technology has been particularly described with reference to thevarious figures and configurations, it should be understood that theseare for illustration purposes only and should not be taken as limitingthe scope of the subject technology.

There may be many other ways to implement the subject technology.Various functions and elements described herein may be partitioneddifferently from those shown without departing from the scope of thesubject technology. Various modifications to these configurations willbe readily apparent to those skilled in the art, and generic principlesdefined herein may be applied to other configurations. Thus, manychanges and modifications may be made to the subject technology, by onehaving ordinary skill in the art, without departing from the scope ofthe subject technology.

It is understood that the specific order or hierarchy of steps in theprocesses disclosed is an illustration of exemplary approaches. Basedupon design preferences, it is understood that the specific order orhierarchy of steps in the processes may be rearranged. Some of the stepsmay be performed simultaneously. The accompanying method claims presentelements of the various steps in a sample order, and are not meant to belimited to the specific order or hierarchy presented.

As used herein, the phrase “at least one of” preceding a series ofitems, with the term “and” or “or” to separate any of the items,modifies the list as a whole, rather than each member of the list (i.e.,each item). The phrase “at least one of” does not require selection ofat least one of each item listed; rather, the phrase allows a meaningthat includes at least one of any one of the items, and/or at least oneof any combination of the items, and/or at least one of each of theitems. By way of example, the phrases “at least one of A, B, and C” or“at least one of A, B, or C” each refer to only A, only B, or only C;any combination of A, B, and C; and/or at least one of each of A, B, andC.

Terms such as “top,” “bottom,” “front,” “rear” and the like as used inthis disclosure should be understood as referring to an arbitrary frameof reference, rather than to the ordinary gravitational frame ofreference. Thus, a top surface, a bottom surface, a front surface, and arear surface may extend upwardly, downwardly, diagonally, orhorizontally in a gravitational frame of reference.

Furthermore, to the extent that the term “include,” “have,” or the likeis used in the description or the claims, such term is intended to beinclusive in a manner similar to the term “comprise” as “comprise” isinterpreted when employed as a transitional word in a claim.

As used herein, the terms “about,” “substantially,” and “approximately”may provide an industry-accepted tolerance for their corresponding termsand/or relativity between items, such as from less than one percent tofive percent.

The word “exemplary” is used herein to mean “serving as an example,instance, or illustration.” Any embodiment described herein as“exemplary” is not necessarily to be construed as preferred oradvantageous over other embodiments.

A reference to an element in the singular is not intended to mean “oneand only one” unless specifically stated, but rather “one or more.”Pronouns in the masculine (e.g., his) include the feminine and neutergender (e.g., her and its) and vice versa. The term “some” refers to oneor more. Underlined and/or italicized headings and subheadings are usedfor convenience only, do not limit the subject technology, and are notreferred to in connection with the interpretation of the description ofthe subject technology. All structural and functional equivalents to theelements of the various configurations described throughout thisdisclosure that are known or later come to be known to those of ordinaryskill in the art are expressly incorporated herein by reference andintended to be encompassed by the subject technology. Moreover, nothingdisclosed herein is intended to be dedicated to the public regardless ofwhether such disclosure is explicitly recited in the above description.

Although the detailed description contains many specifics, these shouldnot be construed as limiting the scope of the subject technology butmerely as illustrating different examples and aspects of the subjecttechnology. It should be appreciated that the scope of the subjecttechnology includes other embodiments not discussed in detail above.Various other modifications, changes and variations may be made in thearrangement, operation and details of the method and apparatus of thesubject technology disclosed herein without departing from the scope ofthe present disclosure. Unless otherwise expressed, reference to anelement in the singular is not intended to mean “one and only one”unless explicitly stated, but rather is meant to mean “one or more.” Inaddition, it is not necessary for a device or method to address everyproblem that is solvable (or possess every advantage that is achievable)by different embodiments of the disclosure in order to be encompassedwithin the scope of the disclosure. The use herein of “can” andderivatives thereof shall be understood in the sense of “possibly” or“optionally” as opposed to an affirmative capability.

What is claimed is:
 1. A method of improving an aesthetic quality of ananatomic feature of a subject, the method comprising administering ahydrogel composition comprising hyaluronic acid, silk fibroin, and an exvivo tissue to the subject, wherein the hyaluronic acid is crosslinkedto the silk fibroin by a water-soluble coupling agent.
 2. The method ofclaim 1, wherein the hydrogel composition is injected into the anatomicfeature of the subject.
 3. The method of claim 1, wherein the hydrogelcomposition is implanted into the anatomic feature of the subject. 4.The method of claim 1, wherein the ex vivo tissue comprises adiposetissue or fat tissue.
 5. The method of claim 1, wherein the ex vivotissue comprises adipose-derived progenitor cells.
 6. The method ofclaim 1, wherein the water-soluble coupling agent comprises a multiaminecrosslinker.
 7. The method of claim 1, wherein the hydrogel compositionhas a weight ratio of the hyaluronic acid to the silk fibroin in therange of about 25:1 to about 1:1.
 8. The method of claim 1, wherein thehyaluronic acid is present in the hydrogel composition in aconcentration of about 16 mg/mL to about 20 mg/mL, and wherein the silkfibroin is present in the hydrogel composition in a concentration ofabout 2 mg/mL to about 5 mg/mL.
 9. The method of claim 1, wherein anappearance of the anatomic feature of the subject is improved.
 10. Themethod of claim 1, wherein a tactile sensation of the anatomic featureof the subject is improved.
 11. The method of claim 1, wherein theanatomic feature of the subject is augmented.
 12. A method of augmentingsoft tissue of a human being comprising: providing a hydrogelcomposition comprising hyaluronic acid and silk fibroin, wherein thehyaluronic acid is crosslinked to the silk fibroin by a water-solublecoupling agent; and mixing the hydrogel composition with an ex vivotissue to produce a hydrogel tissue mixture.
 13. The method of claim 12,further comprising the step of introducing the hydrogel tissue mixtureinto a soft tissue of the human being.
 14. The method of claim 12,wherein the water-soluble coupling agent comprises a multiaminecrosslinker.
 15. The method of claim 14, wherein the multiaminecrosslinker comprises lysine methyl ester.
 16. The method of claim 12,wherein the hydrogel composition has a weight ratio of the hyaluronicacid to the silk fibroin in the range of about 25:1 to about 1:1. 17.The method of claim 12, wherein the hyaluronic acid is present in thehydrogel composition in a concentration of about 16 mg/mL to about 20mg/mL, and wherein the silk fibroin is present in the hydrogelcomposition in a concentration of about 2 mg/mL to about 5 mg/mL. 18.The method of claim 12, wherein the ex vivo tissue comprises adiposetissue or fat tissue.
 19. The method of claim 12, wherein the ex vivotissue comprises adipose-derived progenitor cells.
 20. A method oftreating a soft tissue condition of a subject, the method comprising:administering a hydrogel composition to a site of the soft tissuecondition of the subject, the hydrogel composition comprising hyaluronicacid and silk fibroin crosslinked by a water-soluble coupling agent andan ex vivo adipose tissue, wherein the administration of the hydrogelcomposition improves the soft tissue condition.
 21. The method of claim20, wherein a therapeutically effective amount of the hydrogelcomposition is administered.
 22. The method of claim 20, wherein thehydrogel composition is administered in an amount of from about 0.01 mLto about 200 mL.
 23. The method of claim 20, wherein the hydrogelcomposition is administered using a syringe with a needle or a catheter.24. The method of claim 20, wherein the hydrogel composition isadministered topically.
 25. The method of claim 20, wherein the hydrogelcomposition is administered by direct surgical implantation.
 26. Themethod of claim 20, wherein the hydrogel composition further comprisesan anesthetic.
 27. The method of claim 20, wherein the hydrogelcomposition has a storage modulus of about 1 Pa to about 10,000 Pa. 28.The method of claim 20, wherein the hydrogel composition has a lossmodulus of about 1 Pa to about 500 Pa.
 29. The method of claim 20,wherein the water-soluble coupling agent is a carbodiimide.